EPCAM Antibody, Biotin conjugated

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

EPCAM (Epithelial Cell Adhesion Molecule) is a 40-43 kDa transmembrane glycoprotein expressed on the baso-lateral cell surface in most simple epithelia and a vast majority of carcinomas. The protein acts as a physical homophilic interaction molecule between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) at the mucosal epithelium, providing an immunological barrier as a first line of defense against mucosal infection . EPCAM plays a crucial role in embryonic stem cell proliferation and differentiation, and significantly up-regulates the expression of FABP5, MYC, and cyclins A and E .

The significance of EPCAM as a research target stems from its wide distribution in epithelial tissues and overexpression in various carcinomas. It serves as a valuable marker for distinguishing adenocarcinomas from pleural mesothelioma and hepatocellular carcinoma, as well as serous carcinomas of the ovary from mesothelioma . In human tissues, EPCAM is notably expressed in many regions, including the appendix and colon .

What are the key structural characteristics of biotin-conjugated EPCAM antibodies?

Biotin-conjugated EPCAM antibodies feature several important structural elements:

The biotin conjugation to these antibodies provides enhanced detection sensitivity while maintaining the antibody's binding specificity to EPCAM. The extracellular domain targeting (aa77-202) seen in some clones allows for detection of cell surface EPCAM without cell permeabilization requirements .

How does the choice of immunogen affect biotin-conjugated EPCAM antibody performance?

The immunogen selection is critical for determining antibody specificity and performance. From available research data, biotin-conjugated EPCAM antibodies are generated using several distinct immunogen approaches:

• Recombinant protein fragments: Antibodies like ABIN6158878 use recombinant human EPCAM protein fragments from the extracellular domain (aa77-202) . These antibodies demonstrate excellent specificity for surface EPCAM detection.

• KLH-conjugated synthetic peptides: The bs-1513R-Biotin polyclonal antibody utilizes a KLH-conjugated synthetic peptide derived from human EPCAM (immunogen range 221-314/314) . This approach generates antibodies targeting the C-terminal region.

• Cell preparations containing EPCAM protein: Antibodies like ab79079 (clone VU-1D9) use cell preparations containing EPCAM protein as immunogens . This approach can yield antibodies recognizing native conformational epitopes.

The immunogen choice impacts specificity, cross-reactivity, and application suitability. Extracellular domain-targeted antibodies are particularly valuable for flow cytometry and live cell applications, while antibodies targeting conserved regions might offer better cross-species reactivity .

What are the optimal applications for biotin-conjugated EPCAM antibodies?

Biotin-conjugated EPCAM antibodies demonstrate efficacy across multiple research applications:

For immunohistochemistry applications on paraffin-embedded tissues (IHC-P), biotin-conjugated antibodies like ab79079 have been documented to effectively stain EPCAM at 1-2 μg/mL in human colon carcinoma samples, with distinct cell membrane staining of tumor cells . The biotin conjugation enables signal amplification through avidin/streptavidin systems, enhancing detection sensitivity in tissues with low EPCAM expression.

How do monoclonal and polyclonal biotin-conjugated EPCAM antibodies compare in research applications?

The choice between monoclonal and polyclonal biotin-conjugated EPCAM antibodies depends on research objectives:

Monoclonal Antibodies (e.g., ab79079, ABIN6158878):

• Provide consistent lot-to-lot reproducibility

• Target single epitopes with high specificity

• Clone VU-1D9 (ab79079) is documented for IHC-P applications with minimal background

• Clone EGP40-1372 (ABIN6158878) targets the extracellular domain (aa77-202) and works well in multiple applications including WB, FACS, IF, and IHC

• Recombinant monoclonal EPR20532-225 (ab314940) offers high specificity with reduced background in IHC-P

Polyclonal Antibodies (e.g., bs-1513R-Biotin):

• Recognize multiple epitopes, potentially increasing detection sensitivity

• The bs-1513R-Biotin antibody demonstrates wider species reactivity (human, mouse, rat)

• Suitable for multiple applications (WB, ELISA, IHC-P, IHC-F) with recommended dilutions varying by application

• May provide stronger signals in applications where the target protein is present in low abundance

For applications requiring absolute specificity (e.g., distinguishing specific carcinoma subtypes), monoclonal antibodies are preferable. For maximum sensitivity in detecting EPCAM across multiple species or applications, polyclonal antibodies may offer advantages .

What protocol optimizations enhance biotin-conjugated EPCAM antibody performance in IHC-P?

To optimize IHC-P protocols with biotin-conjugated EPCAM antibodies:

• Antigen retrieval: Heat-induced epitope retrieval methods are recommended, as demonstrated in protocols using Leica BOND™ systems with ab314940 .

• Blocking: Thorough blocking of endogenous biotin is critical, particularly in biotin-rich tissues like liver, kidney, and brain. Use commercial biotin-blocking kits before antibody application.

• Antibody dilution: Optimal dilution varies by product:

  • For ab79079: 1-2 μg/mL shows optimal membrane staining in colon carcinoma

  • For bs-1513R-Biotin: 1:200-400 dilution is recommended

• Detection systems: Streptavidin-HRP systems with DAB chromogen demonstrate excellent results, as seen in published applications of ab79079 .

• Controls: Include known positive control tissues (colon carcinoma) and negative controls (normal skeletal muscle showing negative staining with ab314940) .

• Incubation conditions: Overnight incubation at 4°C may enhance specific binding while reducing background compared to shorter incubations at room temperature.

How can non-specific background be reduced when using biotin-conjugated EPCAM antibodies?

Non-specific background is a common challenge with biotin-conjugated antibodies. Research data suggests several effective strategies:

• Block endogenous biotin: Use commercial avidin/biotin blocking kits before applying biotinylated antibodies, especially for biotin-rich tissues.

• Optimize antibody concentration: Excessive antibody concentration can cause high background. For example, when using bs-1513R-Biotin, dilutions of 1:200-400 for IHC-P provide optimal signal-to-noise ratio .

• Avoid blue fluorescent dyes: If performing fluorescent detection after using biotin-conjugated antibodies, avoid blue fluorescent dyes like CF®405S and CF®405M as they can give higher non-specific background than other dye colors .

• Pre-absorb antibodies: For polyclonal antibodies like bs-1513R-Biotin, pre-absorption against non-relevant tissues may reduce cross-reactivity.

• Select proper negative control tissues: Normal skeletal muscle has been documented as an effective negative control for EPCAM staining, as demonstrated with ab314940 .

• Use TBS buffer with BSA: Storage and dilution in TBS (pH 7.4) with 1% BSA can reduce non-specific binding, as used in the formulation of bs-1513R-Biotin .

What factors influence the cross-species reactivity of biotin-conjugated EPCAM antibodies?

Cross-species reactivity is an important consideration for researchers working with multiple model systems. The available data indicates:

• Epitope conservation: The degree of sequence homology in the target epitope region across species determines cross-reactivity. EPCAM gene orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

• Antibody selectivity table:

• Application influence: Cross-reactivity may vary by application; an antibody may cross-react in Western blot but not in IHC due to differences in protein conformation and epitope accessibility.

• Validation requirement: Even when cross-reactivity is claimed, researchers should validate in their specific experimental system, as manufacturers may predict cross-reactivity based on sequence homology rather than experimental verification .

What are the stability and storage considerations for biotin-conjugated EPCAM antibodies?

Proper storage is essential for maintaining the performance of biotin-conjugated EPCAM antibodies:

• Storage temperature: Most biotin-conjugated EPCAM antibodies should be stored at -20°C for long-term stability, as specified for bs-1513R-Biotin with a recommended storage period of 12 months .

• Buffer composition: Optimal storage buffers typically contain:

  • TBS (pH 7.4)

  • 1% BSA as a stabilizer

  • 0.03% Proclin300 as a preservative

  • 50% Glycerol to prevent freeze-thaw damage

• Aliquoting: Dividing antibodies into single-use aliquots prevents repeated freeze-thaw cycles that can degrade both the antibody and the biotin conjugate.

• Avoid exposure to light: Biotin conjugates may be sensitive to prolonged light exposure, particularly if used in fluorescent detection systems.

• Working solution stability: Diluted antibodies typically maintain reactivity for 1-2 weeks when stored at 4°C, but optimal performance is achieved with freshly diluted solutions.

• Performance monitoring: Include positive controls in each experiment to monitor potential degradation of antibody performance over time.

How can biotin-conjugated EPCAM antibodies advance cancer research?

Biotin-conjugated EPCAM antibodies offer several valuable applications in cancer research:

• Carcinoma differentiation: These antibodies have been specifically documented to distinguish adenocarcinoma from pleural mesothelioma and hepatocellular carcinoma, as well as serous carcinomas of the ovary from mesothelioma .

• Tumor cell identification: The membrane staining pattern observed with antibodies like ab79079 in human colon carcinoma enables precise identification of tumor cells .

• Circulating tumor cell detection: Biotin-conjugated EPCAM antibodies can be used in flow cytometry (like ABIN6158878) for detection and isolation of circulating tumor cells, which often maintain epithelial characteristics .

• Cancer stem cell research: Given EPCAM's role in embryonic stem cell proliferation and differentiation, and its upregulation of MYC and cyclins A and E, these antibodies are valuable for cancer stem cell studies .

• Multiplexed imaging: The biotin conjugation allows for signal amplification and multiplexed imaging protocols where EPCAM can be detected alongside other cancer markers.

• Therapeutic target validation: As EPCAM is being explored as a therapeutic target, these antibodies help validate its expression in patient-derived xenografts and other preclinical models.

What methodological approaches enable multiplexed analysis using biotin-conjugated EPCAM antibodies?

Multiplexed analysis with biotin-conjugated EPCAM antibodies can be achieved through several methodological approaches:

• Sequential multiplexing: Using biotin-conjugated EPCAM antibodies in conjunction with directly labeled antibodies against other targets:

  • First detect EPCAM using avidin/streptavidin systems with one chromogen

  • Follow with directly labeled antibodies for other markers with distinct chromogens

  • This approach works well with antibodies like ab79079 and ab314940 in IHC-P applications

• Multispectral imaging: Combining biotin-conjugated EPCAM antibodies with fluorescent streptavidin conjugates:

  • ABIN6158878 has been documented for immunofluorescence applications

  • Can be combined with directly labeled antibodies in different fluorescent channels

  • Allows for colocalization studies of EPCAM with other cellular markers

• Flow cytometry multiplexing:

  • ABIN6158878 is suitable for flow cytometry applications

  • Can be detected with streptavidin conjugated to fluorophores spectrally distinct from those used for other markers

  • Enables characterization of complex cell populations based on EPCAM and other markers

• Mass cytometry approaches:

  • Biotin-conjugated EPCAM antibodies can be detected with metal-tagged streptavidin

  • Allows integration into high-dimensional CyTOF panels

  • Permits simultaneous analysis of dozens of markers including EPCAM

How does EPCAM antibody detection compare across different tissue types and disease states?

EPCAM expression and antibody detection efficiency show notable variations across tissues and disease states:

The membranous localization of EPCAM necessitates proper tissue preparation and staining techniques. For paraffin sections, heat-induced epitope retrieval is critical for optimal detection, as demonstrated in protocols for ab314940 . Disease states often alter EPCAM expression levels, with many carcinomas showing upregulation compared to their tissues of origin, making biotin-conjugated EPCAM antibodies valuable diagnostic tools.

How can biotin-conjugated EPCAM antibodies be utilized in single-cell analysis technologies?

Biotin-conjugated EPCAM antibodies offer significant advantages in emerging single-cell analysis platforms:

• Single-cell sorting:

  • Biotin-conjugated antibodies like ABIN6158878, suitable for flow cytometry, can be used with streptavidin-fluorophore conjugates for isolation of EPCAM-positive cells for downstream single-cell analysis

  • The signal amplification provided by the biotin-streptavidin system enhances detection of cells with low EPCAM expression

• Single-cell RNA-seq integration:

  • EPCAM-positive cells isolated using biotin-conjugated antibodies can be processed for single-cell transcriptomics

  • This approach enables correlation of EPCAM protein expression with transcriptional profiles at single-cell resolution

  • Particularly valuable for heterogeneity studies in epithelial cancers

• Spatial transcriptomics:

  • Biotin-conjugated EPCAM antibodies can be used in spatial proteogenomic platforms

  • Allows simultaneous visualization of EPCAM protein expression and spatial gene expression patterns

  • Provides insights into tumor microenvironment interactions

• CITE-seq approaches:

  • Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) can incorporate biotin-conjugated EPCAM antibodies

  • Enables simultaneous protein and RNA analysis from the same single cells

  • Helps define epithelial cell states across developmental processes and disease progression

What are the current limitations of biotin-conjugated EPCAM antibodies and potential solutions?

Despite their utility, biotin-conjugated EPCAM antibodies face several limitations that researchers must address:

• Endogenous biotin interference:

  • Problem: Tissues with high endogenous biotin (liver, kidney, brain) can produce false-positive signals

  • Solution: Implement dedicated avidin/biotin blocking steps before antibody application

  • Evidence: Negative control staining in skeletal muscle with ab314940 demonstrates proper blocking efficacy

• Background with certain detection methods:

  • Problem: Blue fluorescent dyes (CF®405S and CF®405M) can give higher non-specific background with biotin-conjugated antibodies

  • Solution: Select alternative fluorophores for detection of low-abundance targets

  • Evidence: Manufacturer recommendations for ABIN6158878 specifically caution against these dye combinations

• Clone-specific variability:

  • Problem: Different clones show variable performance across applications and species

  • Solution: Benchmark multiple clones for specific research applications

  • Evidence: Clone variations are evident when comparing VU-1D9, EPR20532-225, and EGP40-1372

• Conjugation ratio inconsistency:

  • Problem: Batch-to-batch variation in biotin:antibody ratios can affect performance

  • Solution: Titrate each new lot before use in critical experiments

  • Evidence: Recommended dilution ranges (e.g., 1:200-400 for bs-1513R-Biotin) indicate optimization requirements

• Limited multiplexing capacity:

  • Problem: Streptavidin-based detection limits use of multiple biotin-conjugated antibodies simultaneously

  • Solution: Combine with directly labeled antibodies or sequential staining protocols

  • Evidence: Successful implementation in immunohistochemistry applications

What novel research directions are emerging for EPCAM detection beyond traditional antibody applications?

Innovative approaches for EPCAM detection are expanding beyond conventional antibody applications:

• Aptamer-based detection:

  • DNA/RNA aptamers against EPCAM can be biotinylated for detection

  • Offers advantages in stability, production consistency, and smaller size

  • Potential for improved tissue penetration in thick sections

• Nanobody development:

  • Single-domain antibody fragments against EPCAM extracellular domain

  • Smaller size (approximately 15 kDa) compared to conventional antibodies

  • Enhanced tissue penetration while maintaining specificity

  • Can be biotin-conjugated for similar detection approaches

• CRISPR-based detection systems:

  • CRISPR-Cas systems modified for protein detection rather than DNA targeting

  • Potential for programmable recognition of EPCAM epitopes

  • May offer greater specificity for distinguishing closely related isoforms

• Mass spectroscopy imaging integration:

  • Biotin-conjugated EPCAM antibodies detected with metal-tagged streptavidin

  • Allows spatial mapping of EPCAM in tissues using mass spectrometry imaging

  • Enables quantitative analysis of expression levels across tissue regions

• Theranostic applications:

  • Dual-purpose biotin-conjugated antibodies for both imaging and therapeutic delivery

  • Detection of EPCAM combined with targeted delivery of therapeutic payloads

  • Particularly relevant given EPCAM's upregulation in many carcinomas