CEACAM5 Antibody, HRP conjugated

What is CEACAM5 and why is it a significant target for cancer research?

CEACAM5 (Carcinoembryonic Antigen-related Cell Adhesion Molecule 5), also known as CD66e or CEA, is a glycosylated cell surface protein with 702 amino acid residues and a mass of approximately 76.8 kDa. It is primarily localized to the cell membrane . CEACAM5 is rarely expressed in normal adult tissues but is overexpressed in diverse cancers, playing significant roles in tumorigenesis, progression, and metastasis . Its differential expression pattern makes it an ideal target for cancer diagnostics and therapeutics. The protein belongs to the CEA family and is involved in cell adhesion and apoptotic pathways . Research interest in CEACAM5 has intensified due to its potential as a target for antibody-drug conjugates (ADCs) in treating solid tumors .

What are the primary applications for HRP-conjugated CEACAM5 antibodies in research?

HRP-conjugated CEACAM5 antibodies serve multiple research purposes across various experimental techniques:

These applications leverage the specificity of the anti-CEACAM5 antibody combined with the signal amplification provided by the HRP enzyme conjugate, which catalyzes a colorimetric, chemiluminescent, or fluorescent reaction depending on the substrate used .

What tissue expression patterns are expected when using CEACAM5 antibodies?

When using CEACAM5 antibodies for tissue analysis, researchers should expect specific expression patterns. CEACAM5 is normally expressed in columnar epithelial and goblet cells of the colon . In pathological conditions, CEACAM5 shows overexpression in various cancer types including gastric cancer (e.g., MKN-45 cell line), pancreatic carcinoma (e.g., BxPC-3 cell line), and colorectal cancer (e.g., LS174T cell line) . When designing experiments, it's essential to include appropriate positive and negative control tissues to verify antibody specificity and optimize staining protocols.

How should I optimize Western blot protocols using HRP-conjugated CEACAM5 antibodies?

Western blot optimization for HRP-conjugated CEACAM5 antibodies requires attention to several critical parameters:

When troubleshooting, a titration of antibody concentrations may be necessary to determine the optimal signal-to-noise ratio for your specific samples.

What controls should I include when using HRP-conjugated CEACAM5 antibodies in immunohistochemistry?

Rigorous control implementation is essential for reliable IHC results with HRP-conjugated CEACAM5 antibodies:

• Positive tissue control: Include known CEACAM5-expressing tissue such as colon cancer sections or cell blocks from CEACAM5-positive cell lines (e.g., MKN-45, BxPC-3, or LS174T) .

• Negative tissue control: Normal tissues with minimal CEACAM5 expression (e.g., skeletal muscle) serve as specificity controls.

• Isotype control: Use matched isotype HRP-conjugated antibody at the same concentration to assess non-specific binding.

• Antibody omission control: Process tissue sections without primary antibody to evaluate endogenous peroxidase activity and non-specific binding of detection reagents.

• Absorption control: Pre-incubate the antibody with purified CEACAM5 protein to confirm specificity.

• Cell line validation: Use cell lines with known CEACAM5 expression levels (high, moderate, and negative) to validate staining patterns.

• Internal control: Identify tissues with cells that should be both positive and negative within the same section to verify specific staining.

These controls help differentiate true positive signals from artifacts and provide confidence in experimental results, especially when evaluating novel therapeutic approaches targeting CEACAM5 .

What is the optimal dilution range for HRP-conjugated CEACAM5 antibodies across different applications?

Optimal dilution factors vary by application and specific antibody characteristics:

While these ranges provide a starting point, optimization is necessary for each specific antibody and experimental condition. Begin with the manufacturer's recommended dilution and perform a titration series to determine the concentration that yields the highest signal-to-noise ratio for your particular samples .

How can I validate the specificity of HRP-conjugated CEACAM5 antibodies across different cancer cell lines?

Comprehensive validation of CEACAM5 antibody specificity requires multiple complementary approaches:

• Multi-cell line validation: Test the antibody across cell lines with varying CEACAM5 expression levels, including:

  • High expressors: LS174T (colorectal), MKN-45 (gastric), BxPC-3 (pancreatic)

  • Moderate expressors: HT29, Caco-2 (colorectal)

  • Non-expressors: Lymphocyte cell lines (negative control)

• Correlation with mRNA levels: Compare antibody staining intensity with CEACAM5 mRNA levels determined by qRT-PCR.

• siRNA knockdown: Demonstrate reduced staining in cells treated with CEACAM5-specific siRNA compared to scrambled control.

• Recombinant protein competition: Pre-incubate antibody with purified CEACAM5 protein before staining to demonstrate signal reduction.

• Multiple antibody concordance: Compare staining patterns with other validated CEACAM5 antibodies targeting different epitopes.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein.

• CRISPR/Cas9 knockout: Generate CEACAM5 knockout cell lines as definitive negative controls.

This multi-faceted approach helps establish antibody reliability for critical applications, particularly when evaluating novel therapeutic strategies like antibody-drug conjugates targeting CEACAM5 .

What are the optimal fixation and antigen retrieval methods for CEACAM5 detection in FFPE tissue samples?

Optimizing fixation and antigen retrieval is critical for successful CEACAM5 detection in FFPE samples:

For glycosylated membrane proteins like CEACAM5, overfixation can mask epitopes. If using formalin fixation, limit to 24-48 hours. HIER using pressure cooking or microwave heating generally provides better antigen recovery than water bath methods.

Always perform parallel optimization experiments with identical tissue samples to determine the most effective method for your specific antibody and tissue type .

How can I overcome potential cross-reactivity with other CEACAM family members?

CEACAM5 belongs to a family of related adhesion molecules with sequence homology, making cross-reactivity a significant concern:

• Epitope selection: Use antibodies targeting unique CEACAM5 regions with minimal homology to other family members. The N-domain of CEACAM5 shows greater sequence divergence.

• Antibody validation: Test against recombinant proteins or cell lines expressing individual CEACAM family members (CEACAM1, CEACAM3, CEACAM6, CEACAM7, and CEACAM8).

• Pre-absorption: Pre-incubate the antibody with recombinant proteins of related CEACAM family members to reduce cross-reactivity.

• Western blot confirmation: CEACAM family members have different molecular weights - CEACAM5 is 76.8 kDa while others range from 30-90 kDa. Western blot can help confirm target specificity .

• Knockout/knockdown controls: Use genetic manipulation to create CEACAM5-specific knockout/knockdown models while maintaining expression of other family members.

• Alternative detection methods: Confirm results using nucleic acid-based techniques (qPCR, RNA-seq) that can differentiate between family members with high specificity.

• Multiplexed analysis: Use antibodies to multiple CEACAM family members simultaneously with distinct labels to assess relative expression patterns.

Researchers should be particularly cautious about cross-reactivity with CEACAM6, which shares significant homology with CEACAM5 and is often co-expressed in the same tissues .

How can I troubleshoot weak or absent signal when using HRP-conjugated CEACAM5 antibodies?

When facing detection challenges with HRP-conjugated CEACAM5 antibodies, consider these systematic troubleshooting approaches:

For CEACAM5 specifically:

• Glycosylation interference: CEACAM5 is heavily glycosylated, which may mask epitopes. Try treating samples with PNGase F before detection.

• Membrane protein solubilization: Ensure adequate membrane protein extraction using appropriate detergents (e.g., NP-40, Triton X-100).

• Fresh tissue samples: For IHC, use recently cut sections as antigen reactivity can decrease in stored slides.

• Post-conjugation storage: HRP-conjugated antibodies should be stored with stabilizers like 50% glycerol at -20°C, avoiding repeated freeze-thaw cycles.

• Substrate selection: For challenging samples, switch to a more sensitive substrate system (enhanced chemiluminescence or amplified detection) .

What are the optimal blocking reagents to reduce background when using HRP-conjugated CEACAM5 antibodies?

Background reduction requires careful selection of blocking reagents based on the specific application:

For CEACAM5 specifically:

• Endogenous peroxidase quenching: For tissue sections, pre-treat with 3% hydrogen peroxide in methanol for 10 minutes before blocking.

• Endogenous biotin blocking: If using avidin-biotin detection systems, block endogenous biotin with commercial kits.

• Fc receptor blocking: For cell preparations, include 10% serum from the same species as the antibody or use commercial Fc receptor blockers.

• Cross-adsorbed blocking reagents: Use blocking reagents that have been cross-adsorbed against the species of your samples.

• Detergent addition: Include 0.1-0.3% Triton X-100 or 0.05% Tween-20 in blocking solutions to reduce hydrophobic interactions.

Optimal blocking conditions should be determined empirically for each specific antibody and sample type .

How can I develop a sandwich ELISA using HRP-conjugated CEACAM5 antibodies?

Developing a robust sandwich ELISA for CEACAM5 detection requires careful optimization of multiple parameters:

• Capture antibody selection: Choose an antibody targeting an epitope distinct from that of the HRP-conjugated detection antibody. Optimal coating concentration is typically 1-5 μg/mL in carbonate/bicarbonate buffer (pH 9.6).

• Plate coating: Incubate high-binding polystyrene plates with capture antibody overnight at 4°C, followed by 3x washes with PBS-T (PBS + 0.05% Tween-20).

• Blocking: Block remaining binding sites with 1-3% BSA or 5% non-fat dry milk in PBS for 1-2 hours at room temperature.

• Sample preparation: Optimize sample dilution in sample buffer (typically PBS with 0.5-1% BSA and 0.05% Tween-20). For cell culture supernatants, use directly or diluted 1:2 to 1:10. For serum/plasma, typical dilutions range from 1:10 to 1:100.

• Standard curve: Prepare a 7-point standard curve using recombinant CEACAM5 protein with 2-fold serial dilutions, starting at approximately 1000 ng/mL.

• Detection antibody: Use HRP-conjugated anti-CEACAM5 at 0.5-2 μg/mL, incubating for 1-2 hours at room temperature.

• Signal development: Add TMB substrate and incubate for 15-30 minutes protected from light. Stop the reaction with 2N H₂SO₄ and read absorbance at 450 nm with a reference at 570 nm.

• Validation: Determine assay parameters including:

  • Detection limit (typically 0.1-1 ng/mL for optimized ELISAs)

  • Linear range (usually spanning 2 orders of magnitude)

  • Precision (intra- and inter-assay CV should be <10% and <15%, respectively)

  • Recovery (80-120% in spiked samples)

  • Parallelism (serial dilutions should yield consistent calculated concentrations)

For measuring CEACAM5 in clinical samples, special considerations include potential hook effects at high concentrations and interference from heterophilic antibodies, which can be mitigated with appropriate sample diluent additives .

How can HRP-conjugated CEACAM5 antibodies be utilized in cancer biomarker studies?

HRP-conjugated CEACAM5 antibodies offer versatile applications in cancer biomarker research:

• Tissue microarray (TMA) analysis: Enable high-throughput screening of CEACAM5 expression across multiple tumor types and stages. This approach can identify cancer subtypes with differential CEACAM5 expression patterns relevant for targeted therapy.

• Circulating tumor cell (CTC) detection: CEACAM5 antibodies can be used in microfluidic or immunomagnetic CTC isolation systems, followed by on-chip HRP-based visualization.

• Liquid biopsy development: Detection of shed CEACAM5 in serum samples using high-sensitivity ELISA with HRP-conjugated antibodies can serve as a minimally invasive monitoring tool.

• Prognostic biomarker validation: Standardized IHC protocols using HRP-conjugated CEACAM5 antibodies can help establish correlations between expression levels and clinical outcomes across large patient cohorts.

• Therapy response prediction: Serial measurements of CEACAM5 expression before and during treatment can identify expression changes that correlate with therapeutic response.

• Companion diagnostic development: HRP-based CEACAM5 detection can be optimized as a companion diagnostic for CEACAM5-targeted therapies such as antibody-drug conjugates .

• Multiplexed biomarker panels: Combined with antibodies against other cancer markers, CEACAM5 detection can contribute to comprehensive tumor profiling and patient stratification.

When developing such applications, researchers should establish standardized protocols with appropriate controls to ensure reproducibility across different laboratories and clinical settings .

What are the emerging applications of CEACAM5 antibodies in targeted cancer therapies?

CEACAM5 antibodies are finding increasing utility in targeted therapy development:

• Antibody-drug conjugates (ADCs): CEACAM5-targeting antibodies conjugated to cytotoxic payloads like monomethyl auristatin E (MMAE) have shown promising antitumor efficacy. For example, a recent study demonstrated that a single-domain antibody B9 conjugated to MMAE exhibited potent activity against CEACAM5-expressing gastric cancer (MKN-45), pancreatic carcinoma (BxPC-3), and colorectal cancer (LS174T) cell lines with IC₅₀ values of 38.14, 25.60, and 101.4 nM, respectively .

• Radioimmunotherapy: CEACAM5 antibodies labeled with therapeutic radioisotopes (e.g., ¹⁷⁷Lu, ⁹⁰Y) can deliver targeted radiation to tumor sites.

• Bispecific antibodies: Dual-targeting antibodies linking CEACAM5 recognition with T-cell engagement show promise for directing immune responses against CEACAM5-positive tumors.

• CAR-T cell therapy: CEACAM5-specific chimeric antigen receptors are being developed to redirect T cells against CEACAM5-expressing tumors.

• Immunomodulatory antibodies: Some CEACAM5-targeting antibodies may disrupt tumor-promoting signaling pathways independent of payload delivery.

• Small-format antibody derivatives: Single-domain antibodies (like the B9 antibody mentioned in the search results) offer advantages in tumor penetration and reduced immunogenicity compared to conventional antibodies .

• Nanoparticle conjugation: CEACAM5 antibodies conjugated to nanoparticles can deliver diverse therapeutic payloads with improved pharmacokinetics.

Researchers investigating these approaches should carefully consider target expression heterogeneity across tumor cells and potential escape mechanisms when designing preclinical and clinical studies .

What considerations are important when using HRP-conjugated antibodies in multiplexed immunoassays with CEACAM5?

Multiplexed detection involving HRP-conjugated CEACAM5 antibodies requires careful planning:

• Signal separation strategies:

  • Sequential detection with HRP inactivation between rounds

  • Spatial separation using microarray or microfluidic platforms

  • Spectral separation using different chromogenic substrates

  • Tyramide signal amplification with different fluorophores

• Antibody compatibility:

  • Select antibodies raised in different host species to avoid cross-reactivity

  • Verify that epitopes are spatially distinct when using multiple antibodies against CEACAM5

  • Test for potential steric hindrance between antibodies targeting closely positioned epitopes

• Cross-reactivity mitigation:

  • Pre-adsorb antibodies against common cross-reactive proteins

  • Use highly specific monoclonal antibodies rather than polyclonals

  • Include appropriate blocking steps between detection rounds

• Signal balancing:

  • Adjust antibody concentrations to achieve comparable signal intensities

  • Carefully time substrate development for consistent signal generation

  • Use digital image analysis to compensate for signal differences

• Technical considerations:

  • When combining HRP with other enzymes (e.g., alkaline phosphatase), ensure substrate and buffer compatibility

  • For fluorescent multiplex assays, use appropriate filters to prevent bleed-through

  • Consider automated staining platforms for improved reproducibility

• Validation requirements:

  • Run single-marker controls alongside multiplexed assays

  • Include internal reference markers for normalization

  • Verify multiplex results with orthogonal single-marker techniques

These considerations are particularly important when developing comprehensive tumor profiling assays that combine CEACAM5 with other diagnostic or prognostic markers .