EPCAM Antibody, FITC conjugated

What is EPCAM and why is it a significant research target?

EPCAM (Epithelial Cell Adhesion Molecule) is a transmembrane glycoprotein frequently overexpressed in various carcinomas, making it an important biomarker in cancer research . It functions as a homophilic interaction molecule between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) at the mucosal epithelium, providing an immunological barrier against mucosal infection . EPCAM also plays roles in embryonic stem cell proliferation and differentiation, while upregulating the expression of FABP5, MYC, and cyclins A and E . These diverse functions make EPCAM antibodies valuable tools for investigating epithelial tissues, tumor detection, and immune system interactions.

What are the specific characteristics of the VU-1D9 clone EPCAM antibody?

The VU-1D9 clone is a mouse monoclonal antibody (IgG1 isotype) that recognizes an extracellular epitope within the EGF-like domain I of CD326/EPCAM . This antibody has demonstrated high specificity for human EPCAM and strongly stains various normal epithelial cells and carcinomas . The VU-1D9 clone was originally developed using the small cell lung carcinoma cell line H69 as an immunogen . When conjugated to FITC, the purified antibody undergoes optimization to ensure that unconjugated antibody and free fluorochrome are removed by size-exclusion chromatography . Unlike some other anti-EPCAM antibodies such as C215, VU-1D9 binds to a distinct epitope region, as demonstrated in competitive binding assays .

What is the difference between FITC-conjugated and unconjugated EPCAM antibodies?

FITC-conjugated EPCAM antibodies have fluorescein isothiocyanate directly attached to the antibody molecule, allowing for direct detection in flow cytometry and fluorescence microscopy without the need for secondary antibodies . The conjugation process typically involves purifying the antibody and then attaching FITC under optimized conditions, followed by size-exclusion chromatography to remove unconjugated antibody and free fluorochrome .

In contrast, unconjugated EPCAM antibodies require a fluorescently-labeled secondary antibody for detection in flow cytometry and fluorescence microscopy applications. While unconjugated antibodies offer flexibility in detection strategies and potential signal amplification through secondary antibodies, FITC-conjugated antibodies simplify protocols, reduce background, and allow for multiplex staining with other antibodies of different isotypes.

How should I optimize flow cytometry protocols when using FITC-conjugated EPCAM antibodies?

For optimal flow cytometry results with FITC-conjugated EPCAM antibodies, consider the following methodological approach:

• Cell preparation: Harvest 2 × 10^5 target cells and wash with FACS buffer (PBS supplemented with 1% FCS and 0.1% sodium azide) .

• Antibody concentration optimization: Titrate the antibody to determine optimal concentration. Published protocols typically use 2.5 μg/ml of FITC-labeled antibody, but this may vary depending on your specific cell line .

• Incubation conditions: Incubate cells with the antibody for 30 minutes at 4°C in the dark to prevent photobleaching of FITC .

• Washing steps: After incubation, wash cells three times with FACS buffer to reduce background signal.

• Controls: Always include:

  • An isotype control (mouse IgG2a kappa isotype for VU-1D9) to assess non-specific binding

  • Unstained cells to establish autofluorescence baseline

  • Positive control using a known EPCAM-expressing cell line (e.g., HCT-8, HCT116, or SW480)

• FITC compensation: When performing multicolor flow cytometry, properly compensate for FITC spectral overlap with other fluorophores.

• Analysis: Analyze results by comparing the mean fluorescence intensity (MFI) relative to the isotype control .

What are the recommended protocols for validating EPCAM antibody specificity?

Validating EPCAM antibody specificity requires a multi-faceted approach:

• Competitive binding assays: Preincubate target cells with unlabeled EPCAM-specific antibodies (e.g., C215) before adding FITC-labeled EPCAM antibody. If binding is specific to the same epitope, the unlabeled antibody will block binding of the FITC-conjugated antibody, resulting in decreased MFI .

• Cell line validation: Compare binding to:

  • EPCAM-positive cell lines (e.g., HCT-8, Caco-2, HCT116, SW480)

  • EPCAM-negative cell lines or control cells (e.g., CHO-K1 lacking EPCAM expression)

  • EPCAM-transfected cell lines (e.g., HEK293 cells stably or transiently transfected with EPCAM expression vectors)

• Mutant analysis: Test antibody binding to EPCAM glycosylation mutants or domain-specific mutants to confirm epitope specificity .

• Immunohistochemistry cross-validation: Compare flow cytometry results with immunohistochemistry on fixed and unfixed samples to confirm recognition of the same antigen in different contexts .

• Western blotting: Confirm antibody reactivity to denatured EPCAM protein to determine if the epitope is conformational or linear .

How do different anti-EPCAM antibody clones differ in epitope recognition?

Different anti-EPCAM antibody clones recognize distinct epitopes, which affects their binding properties and applications. Based on the research data:

• VU-1D9 clone: Recognizes an extracellular epitope within EGF-like domain I of EPCAM . In competitive binding assays, VU-1D9 does not compete with the HO-3 antibody, indicating they recognize different epitopes .

• C215 clone: Binds to the EGF-like domain I of EPCAM but at a different site than VU-1D9. Peptide library screening showed that C215 recognizes two peptides at amino acid positions 31-52 and 103-124 . C215 competes with HO-3 for binding, suggesting overlapping epitopes .

• HO-3 clone: Recognizes a conformational epitope with three binding sites on EPCAM. The major binding sites include regions within EGF-like domain I and II (amino acids 49-70, 67-88) and a third site at amino acids 175-196 .

The table below summarizes peptide recognition by different antibody clones:

Understanding these differences is crucial for selecting the appropriate antibody clone for specific experimental applications .

What are the key differences between antibodies targeting the EpCL vs. EpRE regions of EPCAM?

The EpCAM extracellular domain contains two distinct regions: EpCL (amino acids 24-80) and EpRE (amino acids 81-265), which display different properties in antibody generation and application:

• Immunogenicity: Analysis of 377 human anti-EPCAM monoclonal antibodies revealed that antibodies against both regions can be generated, with the mass distribution (22.6% EpCL vs. 77.4% EpRE) correlating closely with the molecular weights of these regions (6.2 kDa for EpCL and 21.2 kDa for EpRE) .

• Native conformation recognition: A significantly higher percentage of EpCL-reactive antibodies (66.3%, 55 of 83) can bind to native EPCAM on cell surfaces compared to EpRE-reactive antibodies (5.5%, 16 of 293) . This suggests that the EpCL domain more efficiently induces antibodies recognizing conformational epitopes presented on the cell surface.

• Application differences: Antibodies targeting these different regions may be more suitable for specific applications:

  • EpCL-targeting antibodies: Better for cell surface detection by flow cytometry and immunocytochemistry on unfixed cells

  • EpRE-targeting antibodies: May be more suitable for applications involving denatured protein (e.g., western blotting)

• Affinity discrepancies: Antibodies may show different binding affinities when tested against recombinant EpEX protein by ELISA versus native EPCAM on cell surfaces. For example, antibodies 3C049 and 3C060 showed low affinity in flow cytometry but strong affinity to EpEX protein in ELISA, likely due to differences in protein folding between recombinant and native forms .

These differences highlight the importance of selecting antibodies targeting the appropriate epitope region based on the intended experimental application.

What are common false-negative results when using FITC-conjugated EPCAM antibodies and how can they be addressed?

False-negative results with FITC-conjugated EPCAM antibodies can occur due to several factors:

• Photobleaching: FITC is relatively susceptible to photobleaching.

  • Solution: Minimize exposure to light during sample preparation and storage. Consider using photoprotective mounting media and process samples in low-light conditions.

• Epitope masking: Some fixation protocols may alter or mask the EPCAM epitope.

  • Solution: Compare results using different fixation methods (unfixed, paraformaldehyde, methanol) to determine optimal conditions. The search results indicate that native conformation analysis using unfixed cells can be crucial for detecting surface EPCAM .

• Low EPCAM expression levels: Some cells express EPCAM at levels below detection limits.

  • Solution: Use signal amplification methods such as tyramide signal amplification or consider alternative, more sensitive detection systems.

• pH sensitivity: FITC fluorescence is pH-sensitive (optimal at pH 8).

  • Solution: Ensure buffers are at optimal pH for FITC fluorescence.

• Incorrect antibody clone for target species: Some clones like VU-1D9 are human-specific and do not cross-react with murine EPCAM .

  • Solution: Verify species reactivity before experiments and select appropriate positive controls.

• Competitive binding: Presence of other antibodies targeting similar epitopes can cause interference.

  • Solution: If using multiple EPCAM antibodies, verify they don't compete for binding as demonstrated in the competitive binding assays between C215 and HO-3 .

How can researchers address background issues when using FITC-conjugated EPCAM antibodies?

High background with FITC-conjugated EPCAM antibodies can negatively impact experimental results. Address this issue with these methodological approaches:

• Optimize blocking: Use 1-5% serum (matched to secondary antibody host if using unconjugated primary) or 1% BSA in PBS to reduce non-specific binding.

• Improve washing steps: Perform multiple wash steps (at least 3) with FACS buffer (PBS supplemented with 1% FCS and 0.1% sodium azide) as described in the literature .

• Address autofluorescence:

  • For tissues: Treat with Sudan Black B (0.1-0.3%) or use commercial autofluorescence reducers

  • For flow cytometry: Implement robust compensation controls and consider using spectral analyzers

• Titrate antibody concentration: Optimal concentration may vary by application and cell type. Start with the recommended 2.5 μg/ml concentration and adjust as needed .

• Purification quality: Ensure high-quality antibody preparations where "unconjugated antibody and free fluorochrome are removed by size-exclusion chromatography" .

• Controls: Always include appropriate controls:

  • Isotype control (mouse IgG1 for VU-1D9)

  • EPCAM-negative cell line

  • Secondary-only control (if using an unconjugated primary antibody in a multi-step protocol)

• Alternative detection strategies: If persistent background issues occur with FITC, consider alternative fluorophores with better signal-to-noise ratios like Alexa Fluor dyes.

How should flow cytometry data from EPCAM-FITC staining be analyzed for cancer stem cell identification?

Cancer stem cell (CSC) identification using EPCAM-FITC antibodies requires careful data analysis approaches:

• Gating strategy:

  • First gate on viable cells using appropriate viability dye

  • Exclude doublets using FSC-H vs. FSC-A plots

  • Gate on EPCAM-positive population using isotype control to set threshold

  • For CSC identification, combine with other stem cell markers (e.g., CD44, CD133)

• Population analysis:

  • Analyze EPCAM expression as a continuous variable rather than simply positive/negative

  • Consider EPCAM-high (EPCAM^high) populations, which often correlate with stem-like properties

  • Use mean fluorescence intensity (MFI) relative to isotype control as a quantitative measure

• Multi-marker approach:

  • CSCs are typically defined by multiple markers

  • Create multi-parameter gates (e.g., EPCAM^high/CD44^+/CD24^-)

  • Consider dimensionality reduction techniques like tSNE or UMAP for complex datasets

• Functional validation:

  • Sort EPCAM-positive populations for functional assays (sphere formation, xenograft tumor initiation)

  • Compare gene expression profiles between EPCAM-positive and negative populations

• Heterogeneity assessment:

  • Analyze EPCAM expression variance within tumor samples

  • Consider comparing primary tumor with metastatic sites or circulating tumor cells

How can researchers use EPCAM antibodies to study epithelial-mesenchymal transition (EMT)?

EPCAM antibodies are valuable tools for studying epithelial-mesenchymal transition (EMT), a critical process in development and cancer progression:

• Dynamic EPCAM expression monitoring:

  • EPCAM is typically downregulated during EMT as cells lose epithelial characteristics

  • Use FITC-conjugated EPCAM antibodies to monitor this downregulation in real-time via flow cytometry

  • Compare EPCAM levels before and after EMT induction (e.g., TGF-β treatment, hypoxia)

• Co-expression analysis with EMT markers:

  • Combine EPCAM-FITC with antibodies against:

    • Epithelial markers (E-cadherin, cytokeratins) expected to correlate with EPCAM

    • Mesenchymal markers (N-cadherin, Vimentin) expected to inversely correlate with EPCAM

    • EMT transcription factors (SNAIL, TWIST, ZEB1/2)

• Cell sorting for molecular analysis:

  • Sort EPCAM-high and EPCAM-low populations for:

    • RNA-seq to compare transcriptional profiles

    • Chromatin accessibility studies to understand epigenetic regulation

    • Protein analysis to identify post-translational modifications

• Functional differences assessment:

  • Compare migration and invasion capabilities of EPCAM-high versus EPCAM-low populations

  • Analyze drug resistance patterns between populations

  • Evaluate differences in tumor-initiating capacity and metastatic potential

• Partial EMT detection:

  • Identify cells in intermediate states (partial EMT) that retain some EPCAM expression while gaining mesenchymal markers

  • These hybrid epithelial/mesenchymal cells often have enhanced plasticity and stem-like properties

By carefully quantifying EPCAM expression levels alongside other epithelial and mesenchymal markers, researchers can gain insights into the complex dynamics and heterogeneity of the EMT process.

How do results from FITC-conjugated EPCAM antibodies in flow cytometry compare with other detection methods?

Different detection methods using EPCAM antibodies can yield varying results due to inherent methodological differences:

• Flow cytometry vs. ELISA:

  • Flow cytometry detects native conformational epitopes on cell surfaces

  • ELISA often uses immobilized proteins that may present different epitopes

  • Research has shown that antibodies like 3C049 and 3C060 can have strong affinity in ELISA but low affinity in flow cytometry, "possibly due to the difference in protein folding of EpEX from the native form of EPCAM and the effect of the fixation of EpEX to the ELISA plate surface"

• Flow cytometry vs. immunocytochemistry (ICC):

  • Generally, intensity correlations exist between these methods as both detect cell surface proteins

  • Studies have shown that "ICC staining intensity had some correlation with FCM signal intensity," with antibody 3C101 showing high affinity in both methods, antibodies 1C008, 3C066, and 3C213 showing moderate affinity, and antibodies 3C166, 3C049, and 3C060 showing low affinity

  • Differences may arise from fixation methods used in ICC that can affect epitope accessibility

• Flow cytometry vs. western blotting:

  • Flow cytometry detects native conformational epitopes

  • Western blotting detects denatured proteins

  • Antibodies recognizing conformational epitopes (like some anti-EPCAM antibodies) may fail in western blotting

  • The screening process for antibodies often involves validation across multiple platforms, as seen with EpMab-16 which was validated by "flow cytometry...immunohistochemistry and western blotting"

• Fixed vs. unfixed samples:

  • Unfixed samples preserve native epitopes but require viable cells

  • The literature emphasizes using "unfixed HCT116 and SW480 cells" for detecting native EPCAM

  • Fixation can mask certain epitopes while revealing others

Understanding these methodological differences is crucial when comparing results across different experimental platforms or when troubleshooting discrepancies in EPCAM detection.

What are the advantages and limitations of using FITC as a conjugate for EPCAM antibodies compared to other fluorophores?

FITC conjugation offers specific advantages and limitations that researchers should consider when selecting fluorophores for EPCAM detection:

Advantages:

• Well-established: FITC is one of the most widely used fluorophores with extensive literature validation, including for anti-EPCAM antibodies like VU-1D9 .

• Bright initial signal: FITC has a high quantum yield, producing bright fluorescence when freshly prepared.

• Cost-effective: Generally less expensive than newer generation fluorophores.

• Spectral compatibility: FITC's excitation/emission profile (495/519 nm) is compatible with standard flow cytometers and fluorescence microscopes.

• Well-defined conjugation chemistry: The isothiocyanate group readily reacts with primary amines on antibodies, allowing for standardized conjugation protocols as described in the literature where "purified antibody is conjugated with fluorescein isothiocyanate (FITC) under optimum conditions" .

Limitations:

• Photobleaching: FITC bleaches relatively quickly compared to newer fluorophores, which can affect long-term imaging or sorting applications.

• pH sensitivity: FITC fluorescence decreases significantly at lower pH values, which can affect results in acidic environments.

• Autofluorescence overlap: FITC emission overlaps with cellular autofluorescence, potentially reducing signal-to-noise ratio, especially in tissues with high intrinsic fluorescence.

• Spectral spillover: In multicolor applications, FITC has substantial spillover into other channels, requiring careful compensation.

• Lower brightness than newer dyes: Newer fluorophores like Alexa Fluor 488 offer greater photostability and brightness.

When selecting between FITC and alternative fluorophores (Alexa Fluor 488, PE, APC) for EPCAM detection, researchers should consider their specific application needs, including required sensitivity, imaging duration, multiplex requirements, and available instrumentation.

How can EPCAM antibodies be used for circulating tumor cell (CTC) detection and characterization?

EPCAM antibodies are crucial tools for CTC research due to the frequent expression of EPCAM on epithelial-derived cancer cells:

• Enrichment strategies:

  • Immunomagnetic separation using EPCAM antibodies conjugated to magnetic beads

  • Microfluidic devices coated with EPCAM antibodies for CTC capture

  • Flow cytometry-based sorting using FITC-conjugated EPCAM antibodies like VU-1D9

• Multi-marker phenotyping:

  • Combine EPCAM-FITC with other markers:

    • Cytokeratins (confirming epithelial origin)

    • CD45 (excluding leukocytes)

    • Mesenchymal markers (Vimentin, N-cadherin) to identify CTCs undergoing EMT

  • This approach addresses the limitation that some CTCs downregulate EPCAM during EMT

• Downstream molecular analysis:

  • Single-cell RNA sequencing of EPCAM-positive CTCs

  • Genomic profiling to identify mutations in CTCs compared to primary tumor

  • Protein analysis to characterize signaling pathway activation

• Functional studies:

  • Viability assessment of isolated CTCs

  • Drug sensitivity testing of EPCAM-positive CTCs

  • Culture and expansion of CTCs for further characterization

• Clinical correlation research:

  • Quantification of EPCAM-positive CTCs as a prognostic biomarker

  • Longitudinal monitoring of CTC phenotypes during treatment

  • Investigation of EPCAM expression heterogeneity in CTCs versus primary tumors

The specificity of antibodies like VU-1D9, which "strongly stains various normal epithelial cells and carcinomas" , makes them valuable for distinguishing epithelial-derived CTCs from other blood components, though researchers must consider potential limitations related to EMT-associated EPCAM downregulation.

What is the role of EPCAM antibodies in investigating cancer immunotherapy approaches?

EPCAM antibodies are increasingly important in cancer immunotherapy research, leveraging EPCAM's frequent overexpression in carcinomas:

• Antibody-dependent cellular cytotoxicity (ADCC) studies:

  • Anti-EPCAM monoclonal antibodies can be evaluated for their ability to induce ADCC

  • Research has focused on "whether these anti-EPCAM mAbs induced antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antitumor activity against CRC in a murine xenograft model"

  • Flow cytometry with FITC-conjugated antibodies can be used to quantify antibody binding to target cells before functional ADCC assays

• Bispecific antibody development:

  • EPCAM-targeting bispecific antibodies that simultaneously engage T cells (e.g., EPCAM × CD3)

  • Characterization of binding domains using epitope mapping techniques, as demonstrated with antibodies like HO-3 and C215

  • Flow cytometry with FITC-conjugated EPCAM antibodies can validate bispecific antibody binding to target cells

• Chimeric antigen receptor (CAR) T-cell therapy:

  • EPCAM as a target for CAR T-cell therapy in epithelial cancers

  • Verification of CAR binding to EPCAM using flow cytometry

  • Optimization of EPCAM-specific single-chain variable fragments (scFvs) derived from antibodies

• Antibody-drug conjugates (ADCs):

  • Development of EPCAM-targeted ADCs for selective delivery of cytotoxic agents

  • Characterization of internalization dynamics of different anti-EPCAM clones

  • Flow cytometry with FITC-conjugated antibodies to assess target expression levels

• Immune checkpoint modulation:

  • Investigation of EPCAM's role in modulating immune responses

  • EPCAM's function "as a physical homophilic interaction molecule between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) at the mucosal epithelium for providing immunological barrier" suggests potential immunomodulatory roles

  • Characterization of immune cell populations in EPCAM-expressing tumor microenvironments

Understanding the epitope specificity and binding characteristics of different EPCAM antibody clones is crucial for these applications, as demonstrated by the detailed epitope mapping studies comparing antibodies like HO-3, C215, and VU-1D9 .

How might novel EPCAM antibody development techniques improve cancer diagnostics and therapeutics?

Emerging techniques in EPCAM antibody development hold promise for advancing cancer research and clinical applications:

• Cell-based immunization and screening (CBIS):

  • This approach, demonstrated in the development of EpMab-16, involves "immunizing one mouse with CHO/EpCAM cells and fusing its spleen cells with P3U1 cells"

  • CBIS ensures antibodies recognize native conformational epitopes on cell surfaces

  • This technique can yield antibodies with superior binding to membrane-bound EPCAM compared to conventional methods using recombinant proteins

• Humanized and fully human antibodies:

  • Development of fully human anti-EPCAM antibodies using technologies like the TC-mAb mice described in the literature

  • These approaches reduce immunogenicity for therapeutic applications

  • Research has demonstrated that "a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis"

• Rational epitope targeting:

  • Using structural data to design antibodies targeting specific functional domains of EPCAM

  • Differential targeting of EpCL vs. EpRE regions based on findings that "the EpCL domain of the EpEX recombinant protein more efficiently induced mAbs that bind to conformational epitopes presented on the cell surface"

  • This approach could yield antibodies with optimized diagnostic or therapeutic properties

• Multi-omics-guided antibody selection:

  • Integrating genomic, transcriptomic, and proteomic data to identify patient-specific EPCAM epitope variants

  • Developing antibody panels targeting different EPCAM epitopes for personalized medicine applications

• Engineered antibody formats:

  • Development of smaller antibody formats (nanobodies, affibodies) targeting EPCAM

  • Creation of multi-specific formats combining EPCAM targeting with other modalities

  • These novel formats may offer improved tissue penetration and reduced immunogenicity

These advanced approaches build upon established techniques such as the production of FITC-conjugated antibodies where "purified antibody is conjugated with fluorescein isothiocyanate (FITC) under optimum conditions" , potentially leading to next-generation tools for both research and clinical applications.

What are the emerging applications of EPCAM antibodies in organoid and 3D culture systems?

EPCAM antibodies are becoming increasingly valuable tools in advanced 3D culture systems and organoid research:

• Organoid establishment and characterization:

  • FITC-conjugated EPCAM antibodies allow non-destructive monitoring of epithelial identity in developing organoids

  • Flow cytometric isolation of EPCAM-positive stem/progenitor cells for organoid initiation

  • Tracking EPCAM expression changes during organoid differentiation and maturation

• Epithelial-stromal interactions in 3D co-cultures:

  • EPCAM antibodies enable visualization of boundaries between epithelial and stromal compartments

  • Investigation of EPCAM's role in "physical homophilic interaction" in complex 3D tissue architectures

  • Analysis of how stromal cells influence EPCAM expression and epithelial organization

• Disease modeling with patient-derived organoids:

  • Comparative analysis of EPCAM expression between normal and diseased organoids

  • Assessment of drug effects on EPCAM-positive populations in tumor organoids

  • Correlation of EPCAM expression patterns with patient tumor characteristics and outcomes

• Developmental biology applications:

  • Studying EPCAM's role in "embryonic stem cells proliferation and differentiation" using organoid models

  • Investigating the dynamics of EPCAM expression during tissue morphogenesis

  • Analysis of EPCAM's functions in regulating "the expression of FABP5, MYC and cyclins A and E" in 3D culture systems

• Organoid-based biobanking and screening:

  • Using EPCAM antibodies to verify epithelial identity and purity of organoid biobanks

  • High-content screening of drug effects on EPCAM-positive cells in organoid systems

  • Development of organoid-based assays for personalized medicine applications

These applications leverage antibody specificity like that of VU-1D9, which "recognizes an extracellular epitope within EGF-like domain I of CD326/EPCAM" , to enable sophisticated analysis of epithelial cells in advanced culture systems that better recapitulate in vivo physiology.