CEACAM8 Recombinant Monoclonal Antibody

What is CEACAM8 and why is it significant in immunological research?

CEACAM8, also known as CD66b, NCA-95, or CD67, is a GPI-anchored glycoprotein with eleven N-glycosylation sites and a molecular weight of approximately 95 kDa. It functions primarily as a cell surface receptor on neutrophils and is essential for facilitating neutrophil adhesion, migration, and transendothelial passage during immune responses. The significance of CEACAM8 lies in its central role in neutrophil recruitment to infection or inflammation sites, where these cells defend against microbial pathogens and contribute to inflammatory responses .

Recent bioinformatics analyses have implicated CEACAM8 in multiple sclerosis pathology during pregnancy, suggesting its potential role in autoimmune conditions. Additionally, CEACAM8 has been investigated as a target in osteomyelitis treatment since 2010, and emerging research positions it as a promising candidate within the CEA family for various cancer studies, particularly those affecting reproductive and digestive systems .

How are CEACAM8 recombinant monoclonal antibodies produced?

CEACAM8 recombinant monoclonal antibodies are produced through a sophisticated in vitro process that enables precise control over antibody characteristics. The production begins with in vitro cloning of genes encoding both heavy and light chains of the CEACAM8 antibody. These genes are then integrated into expression vectors, which are subsequently introduced into host cells. This enables expression of the recombinant antibody within a controlled cell culture environment .

Following expression, the antibodies are purified from the supernatant of transfected host cell lines using affinity chromatography, ensuring high purity and specificity. This recombinant production approach offers several advantages over traditional hybridoma-based methods, including enhanced batch-to-batch consistency, simplified scale-up capability, and guaranteed future supply security .

What are the key applications of CEACAM8 antibodies in research?

CEACAM8 antibodies are versatile tools with multiple research applications:

These applications enable researchers to study CEACAM8 expression, localization, and function in various experimental systems. The antibodies have demonstrated remarkable specificity in binding to human CEACAM8 protein, making them reliable tools for investigating neutrophil biology and CEACAM8-related disease mechanisms .

What validation data should researchers review before selecting a CEACAM8 antibody?

When selecting a CEACAM8 antibody, researchers should carefully review several types of validation data:

• Specificity testing: Verification that the antibody binds to CEACAM8 and not to other related proteins. Look for evidence of testing on both recombinant proteins and endogenous CEACAM8 in appropriate cell types.

• Application-specific validation: Confirmation that the antibody works in your intended application (flow cytometry, Western blot, etc.). For example, search result demonstrates validation in flow cytometry showing that the antibody stains CEACAM8 transfectants but not irrelevant transfectants.

• Cross-reactivity assessment: Documentation of species reactivity. Some antibodies like DF10151 react with both human and rat CEACAM8, while others may be human-specific .

• Control experiments: Data showing appropriate positive and negative controls, such as the comparison between CEACAM8-transfected HEK293 cells versus irrelevant transfectants .

• Protein molecular weight confirmation: Verification that the detected protein matches the expected molecular weight of CEACAM8 (38 kDa calculated or 95 kDa with glycosylation) .

• Batch consistency data: For recombinant antibodies, information about production consistency across batches, which is especially important for longitudinal studies .

What are the optimal protocols for detecting CEACAM8 in human neutrophils?

The detection of CEACAM8 in human neutrophils requires specific protocols optimized for these primary cells:

Flow Cytometry Protocol:

• Isolate fresh human neutrophils using density gradient centrifugation

• Wash cells in PBS containing 2% FBS

• Block Fc receptors with human FcR blocking reagent (10 minutes, 4°C)

• Stain with anti-CEACAM8 PE-conjugated antibody at 1:100 dilution (30 minutes, 4°C, protected from light)

• Wash twice with PBS/2% FBS

• Analyze immediately, as neutrophils have limited viability ex vivo

This approach has been validated with antibodies like the Mouse Anti-Human CEACAM-8/CD66b PE-conjugated Monoclonal Antibody (Clone # 913542), which effectively stains human blood granulocytes . When analyzing results, it's critical to include appropriate isotype controls and to consider that CEACAM8 expression increases significantly upon neutrophil activation.

For multiparameter flow cytometry, CEACAM8 antibodies can be combined with other neutrophil markers (CD15, CD16) and activation markers to comprehensively characterize neutrophil populations and their functional states.

How should researchers troubleshoot weak or non-specific CEACAM8 staining?

When encountering weak or non-specific CEACAM8 staining, researchers should consider the following troubleshooting approaches:

For weak staining:

• Antibody titration: Test a range of antibody concentrations (1:50 to 1:200 for flow cytometry)

• Sample freshness: Ensure neutrophils are freshly isolated, as CEACAM8 expression can decrease in culture

• Fixation protocol adjustment: Optimize fixation conditions, as overfixation can mask epitopes

• Signal amplification: Consider secondary antibody approaches or more sensitive detection systems

• Cell activation status: Remember that resting neutrophils express CEACAM8 in only a limited fraction of the plasma membrane; activation increases surface expression

For non-specific staining:

• Blocking optimization: Increase blocking time/concentration to reduce Fc receptor binding

• Antibody quality check: Verify antibody hasn't degraded; some require storage at -80°C

• Negative controls: Include both isotype controls and CEACAM8-negative cells (e.g., lymphocytes)

• Cross-reactivity testing: Test antibody on cells expressing other CEACAM family members

• Washing protocol adjustment: Increase washing steps or washing buffer stringency

Methodologically, it's important to validate any troubleshooting changes with appropriate controls and to maintain consistent protocols across experiments to ensure reproducibility.

How can researchers effectively use CEACAM8 antibodies in multiplex assays?

Multiplex assays involving CEACAM8 antibodies require careful consideration of compatibility and optimization:

For multiplex flow cytometry:

• Select antibody conjugates with non-overlapping fluorescence spectra

• Validate compensation settings with single-stained controls

• When using PE-conjugated CEACAM8 antibodies (like FAB4246P), ensure minimal spectral overlap with other channels

• Consider the order of staining when combining surface and intracellular markers

For cytometric bead arrays:
Use validated matched antibody pairs, such as the Proteintech MP00293-1 set which includes 83105-3-PBS (capture) and 83105-1-PBS (detection) antibodies specifically validated for cytometric bead array applications . This enables quantitative detection of CEACAM8 alongside other analytes of interest.

For multiplex imaging:

• Use unconjugated antibodies in PBS-only formulations (like 83105-3-PBS) which are ready for custom conjugation

• Perform sequential staining to minimize cross-reactivity

• Include appropriate blocking steps between antibody applications

• Validate spectral unmixing parameters when using multiple fluorophores

Critical methodological consideration: When designing multiplex panels, always include FMO (Fluorescence Minus One) controls to accurately set gates and account for spectral spillover between channels, particularly important when analyzing rare cell populations or subtle expression differences.

How can CEACAM8 antibodies be utilized to investigate neutrophil-tumor cell interactions?

CEACAM8 antibodies offer sophisticated approaches to studying neutrophil-tumor interactions:

Methodology for co-culture experiments:

• Isolate primary neutrophils or use differentiated HL-60 cells

• Label neutrophils with cell tracker dyes

• Co-culture with tumor cell lines (consider 3D culture systems for physiological relevance)

• Use anti-CEACAM8 antibodies to:

  • Block CEACAM8-mediated interactions (functional studies)

  • Monitor neutrophil activation status during co-culture (flow cytometry)

  • Visualize neutrophil-tumor cell contacts (confocal microscopy)

• Quantify neutrophil infiltration, NETosis, and tumor cell killing

For mechanistic studies: Researchers can employ CEACAM8 antibodies alongside inhibitors of specific signaling pathways to dissect the molecular mechanisms of neutrophil-tumor interactions. This approach can reveal whether CEACAM8 signaling synergizes with or antagonizes other pathways in the tumor microenvironment.

CEACAM8's emerging role in cancer research, particularly in reproductive and digestive system tumors , makes it a valuable target for investigating tumor-associated neutrophils (TANs) and their dual pro- and anti-tumorigenic functions. Methodologically, it's crucial to verify antibody specificity in the complex tumor microenvironment where multiple CEACAM family members may be expressed.

What are the considerations for using CEACAM8 antibodies in studies of neutrophil extracellular traps (NETs)?

Using CEACAM8 antibodies in NET studies requires specific methodological considerations:

NET induction and visualization protocol:

• Seed neutrophils on poly-L-lysine coated coverslips

• Stimulate with PMA, LPS, or physiological stimuli

• Fix cells carefully to preserve NET structures

• Perform immunofluorescence staining with:

  • Anti-CEACAM8 antibodies (optimized for IF/ICC applications)

  • DNA stains (DAPI/Hoechst)

  • Additional NET markers (MPO, NE, citrullinated histone H3)

• Image using confocal or super-resolution microscopy

Key methodological considerations:

• Fixation protocol: Standard paraformaldehyde fixation may damage NETs; consider methanol fixation or specialized NET preservation protocols

• Antibody penetration: Ensure antibodies can access epitopes within the NET structure

• Quantification approaches: Develop consistent methods for quantifying CEACAM8 incorporation into NETs

• Live cell imaging: For dynamic studies, consider using non-disruptive concentrations of labeled Fab fragments

Researchers should note that CEACAM8 may be modified during NET formation, potentially affecting antibody recognition. Additionally, comparing CEACAM8 distribution in NETs versus intact neutrophils can provide insights into its role in NET formation and function.

How can researchers quantitatively assess CEACAM8 post-translational modifications using antibody-based approaches?

Quantitative assessment of CEACAM8 post-translational modifications (PTMs) requires sophisticated antibody-based strategies:

Available PTM sites on CEACAM8:

Methodological approaches:

• Immunoprecipitation-Western blot workflow:

  • Immunoprecipitate CEACAM8 using a total CEACAM8 antibody

  • Probe with PTM-specific antibodies

  • Quantify signal ratios between modified and total protein

• Flow cytometry-based approach:

  • Simultaneously stain cells for total CEACAM8 and specific PTMs

  • Calculate PTM/total protein ratios at single-cell level

  • Correlate with functional readouts

• Enzymatic treatments:

  • Treat samples with specific deglycosylases, phosphatases, or deacetylases

  • Compare antibody binding before and after treatment

  • Quantify the contribution of specific modifications to total CEACAM8 signal

For accurate quantitation, researchers should include appropriate controls (e.g., phosphatase inhibitors for phosphorylation studies) and consider the potential impact of one PTM on the detection of others, as modifications can affect epitope accessibility or antibody binding.

What are the latest findings on CEACAM8's role in autoimmune pathologies and how can researchers investigate these mechanisms?

Recent findings suggest CEACAM8 plays significant roles in autoimmune conditions:

Methodological approaches to investigate CEACAM8 in autoimmunity:

• Transcriptional profiling:

  • Compare CEACAM8 expression in patient vs. healthy neutrophils

  • Correlate expression with disease markers and activity scores

  • Use RNA-seq to identify co-regulated genes in CEACAM8 pathways

• Functional neutrophil assays:

  • Assess neutrophil adhesion, migration, and NET formation using CEACAM8 blocking antibodies

  • Compare responses between autoimmune patients and controls

  • Investigate how autoantibodies might affect CEACAM8 function

• Animal models:

  • Use CEACAM8 antibodies to detect infiltrating neutrophils in tissue sections

  • Employ blocking antibodies in disease models to assess functional contributions

  • Consider humanized mouse models for better translation of CEACAM8 biology

• Biomarker development:

  • Evaluate soluble CEACAM8 in patient fluids using matched antibody pairs in ELISA

  • Correlate with disease activity and treatment response

  • Develop multiplex panels including CEACAM8 and other inflammatory markers

When designing these studies, researchers should consider that CEACAM8 may interact with other CEACAM family members to exert regulatory functions , necessitating a systems biology approach to fully understand its role in autoimmune pathologies.