CEACAM4 Antibody

What is CEACAM4 and what is its relationship to other CEACAM family members?

CEACAM4 is a member of the Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) family. It shares structural similarities with CEACAM3, including an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The two proteins display high homology (73% amino acid identity) in their carboxy-terminal intracellular regions, but less sequence identity (49%) in their extracellular Ig V-like domains, particularly in the CFG face of the Ig fold . Unlike several other CEACAM family members, CEACAM4 is primate-specific, with no ortholog present in rodent genomes .

Experimental approach: To study CEACAM4's relationship to other family members, researchers typically use sequence alignment analysis of the different domains and phylogenetic analysis to determine evolutionary relationships. For antibody specificity testing, direct ELISAs can confirm that anti-CEACAM4 antibodies do not cross-react with CEACAM-1, -3, -5, -6, -7, or -8 .

What is the expression pattern of CEACAM4 in human tissues and cell lines?

CEACAM4 is primarily expressed in cells of myeloid lineage. The gene was originally cloned from a pooled human leukocyte cDNA library, suggesting expression in phagocytic cells similar to CEACAM3 . Quantitative RT-PCR analysis has shown that CEACAM4 mRNA increases 6-7 fold during the differentiation of HL60 promyeloid cells toward a granulocyte phenotype when treated with retinoic acid .

Western blot analysis using specific anti-CEACAM4 antibodies has detected the protein in several cell lines, including:

• HL-60 (human acute promyelocytic leukemia)

• THP-1 (human acute monocytic leukemia)

• IMR-90 (human lung fibroblast)

• NCI-H596 (human lung adenosquamous carcinoma)

• Human lung cancer tissue

CEACAM4 typically appears as a band of approximately 30 kDa on Western blots under reducing conditions .

How can researchers study CEACAM4 function in the absence of known physiological ligands?

Studying orphan receptors like CEACAM4 presents significant challenges. One effective approach is to generate chimeric constructs that combine domains from related receptors with known ligands. Researchers have successfully used this method by creating a CEACAM3/4 chimera:

Methodology:

• Create fusion proteins containing the extracellular bacteria-binding domain of CEACAM3 and the transmembrane and cytoplasmic parts of CEACAM4

• Express the chimeric protein in appropriate cell lines (e.g., HEK-293 cells)

• Expose cells to bacteria expressing Opa proteins that bind to the CEACAM3 portion

• Assess phagocytosis using gentamicin protection assays or fluorescence microscopy

• Study signaling events by analyzing tyrosine phosphorylation and recruitment of SH2 domain-containing proteins

This approach allowed researchers to demonstrate that the CEACAM4 cytoplasmic domain becomes tyrosine phosphorylated upon receptor clustering and can support efficient phagocytosis of particulate ligands .

What are the key signaling mechanisms downstream of CEACAM4 activation?

CEACAM4 contains a functional ITAM-like sequence that serves as the basis for its signaling capabilities. Upon receptor clustering:

• Tyrosine residues within the ITAM (positions 222 and 233) become phosphorylated by Src family protein tyrosine kinases (PTKs)

• The phosphorylated ITAM serves as a docking site for SH2 domain-containing proteins including:

  • Src family PTKs

  • Phosphatidylinositol 3-kinase (PI3K)

  • The adapter molecule Nck

• Deletion of the ITAM sequence or inhibition of Src family PTKs with PP2 blocks CEACAM4-mediated phagocytosis

Experimental approach: To study these signaling events, researchers can use GST-pull down assays with GST-SH2 domain fusion proteins, co-immunoprecipitation studies, and peptide spot membranes to identify direct binding partners. Functional experiments using pharmacological inhibitors (e.g., PP2) or expression of mutant constructs (Y222/233F) provide evidence for the role of specific signaling molecules in CEACAM4-mediated functions .

What methods are available for detecting CEACAM4 protein expression?

Detection of CEACAM4 can be challenging due to its restricted expression pattern and potential cross-reactivity with related CEACAM family members. Several approaches are available:

• Western blot analysis:

  • Use of specific monoclonal antibodies (e.g., Clone #568410, MAB4354)

  • Recommended concentration: 2 μg/mL

  • Expected molecular weight: approximately 30 kDa under reducing conditions

  • Suitable for detection in cell lysates from myeloid lineage cells and certain cancer cell lines

• Immunohistochemistry:

  • Monoclonal antibodies raised against recombinant human CEACAM-4 (Phe36-Val140)

  • Validated for formaldehyde-fixed tissues

• Quantitative RT-PCR:

  • TaqMan probe-based assays can discriminate between CEACAM3 and CEACAM4 mRNA

  • Useful for analyzing expression in cell lines and primary cells during differentiation

Validation controls: When using antibodies, confirm specificity against recombinant CEACAM proteins (1-8) and include appropriate positive control cell lines (HL-60, THP-1) and negative controls .

How can researchers generate and validate CEACAM4-specific antibodies?

Generation of specific antibodies against CEACAM4 requires careful design and validation strategies to avoid cross-reactivity with other CEACAM family members:

• Immunogen selection:

  • Use of E. coli-derived recombinant human CEACAM-4 (Phe36-Val140) as an immunogen

  • Consider regions with low homology to other CEACAM family members

  • Focus on the extracellular Ig V-like domain which shows only 49% identity with CEACAM3

• Screening methodology:

  • Direct ELISAs against recombinant CEACAM-1, -3, -4, -5, -6, -7, and -8 to ensure specificity

  • Western blot analysis with cell lines known to express CEACAM4

  • Competition assays with recombinant proteins

• Validation experiments:

  • Test antibody recognition of CEACAM4 in both native and denatured forms

  • Perform siRNA knockdown experiments to confirm specificity

  • Test cross-reactivity with other CEACAM family members in overexpression systems

The most successful approach for generating CEACAM4-specific monoclonal antibodies has been to immunize mice with recombinant protein and screen extensively for clones that do not cross-react with other family members .

What experimental approaches can be used to identify physiological ligands for CEACAM4?

Identifying ligands for orphan receptors like CEACAM4 is challenging but several approaches can be employed:

• Expression cloning strategies:

  • Generate CEACAM4-Fc fusion proteins to use as "bait"

  • Screen cellular or pathogen-derived expression libraries

  • Use flow cytometry or immunoprecipitation to identify interacting partners

• Proximity labeling techniques:

  • Express CEACAM4 fused to promiscuous biotin ligases (BioID or TurboID)

  • Identify proteins in close proximity through streptavidin pulldown and mass spectrometry

• Pathogen binding assays:

  • Screen diverse bacterial and viral strains for binding to CEACAM4

  • Focus on gram-negative bacteria known to interact with other CEACAM family members

  • Use flow cytometry, microscopy, or ELISA-based binding assays

• Cell adhesion and interaction studies:

  • Test whether CEACAM4 mediates homophilic or heterophilic interactions

  • Perform cell aggregation assays with cells expressing CEACAM4

  • Use recombinant soluble CEACAM4 to block potential interactions

The identification of physiological CEACAM4 ligands would be a significant step in understanding this receptor's biology, as current research suggests it may play a primate-specific role in phagocytosis and immune function .

What are the limitations of animal models for studying CEACAM4 function?

CEACAM4 research faces significant challenges regarding animal models:

• Absence in rodent genomes:

  • The CEACAM4 gene is exclusive to the primate lineage

  • Conventional mouse models cannot be used to study CEACAM4 function in vivo

• Methodological alternatives:

  • Humanized mouse models expressing human CEACAM4

  • Primate models (ethical and practical limitations)

  • In vitro systems using human cells

  • Chimeric receptor approaches

• Experimental design considerations:

  • When designing experiments, researchers must account for species-specific differences in CEACAM family composition

  • Studies of CEACAM4 function must rely on human cell lines or primary human cells

  • Results from related receptors in mouse models (e.g., CEACAM1) may not translate directly to human CEACAM4 function

The primate-specific nature of CEACAM4 suggests it may provide unique contributions to the human immune system that cannot be modeled in conventional rodent systems .

How can functional assays be optimized to study CEACAM4-mediated phagocytosis?

Based on the available research, several functional assays can be optimized to study CEACAM4-mediated phagocytosis:

• Gentamicin protection assay:

  • Seed cells (4 × 10^5) in 24-well plates coated with fibronectin (4 μg/ml) and poly-L-lysine (10 μg/ml)

  • Infect with bacteria at a multiplicity of infection (MOI) of 30 bacteria/cell

  • After 1 hour of infection, kill extracellular bacteria with gentamicin (50 μg/ml, 45 min)

  • Lyse cells with 1% saponin in PBS

  • Determine viable internalized bacteria by plating on appropriate media

• Bacterial adherence assay:

  • Follow the same procedure as the gentamicin protection assay but omit the gentamicin treatment

  • Gently wash cells before lysis with 1% saponin

  • Determine total cell-associated bacteria by plating on appropriate media

• Immunofluorescence-based phagocytosis assay:

  • Differential staining of intracellular versus extracellular bacteria

  • Fix cells after infection

  • Label extracellular bacteria with antibodies before permeabilization

  • Label all bacteria after permeabilization with a different fluorophore

  • Intracellular bacteria will be labeled with only one fluorophore

For inhibition studies, pretreat cells with specific inhibitors (e.g., Src inhibitor PP2) 15 minutes before infection to assess the role of particular signaling pathways .

What strategies can be employed to study CEACAM4 interaction with the cytoskeleton during phagocytosis?

Investigating CEACAM4's interaction with the cytoskeleton during phagocytosis can be approached through several complementary methods:

• Microscopy-based approaches:

  • Confocal microscopy to visualize colocalization of CEACAM4 with cytoskeletal elements

  • Live-cell imaging with fluorescently tagged CEACAM4 and cytoskeletal proteins

  • Super-resolution microscopy to examine nanoscale organization at the phagocytic cup

• Biochemical approaches:

  • Co-immunoprecipitation of CEACAM4 with cytoskeletal proteins

  • GST-pull down assays with the cytoplasmic domain of CEACAM4

  • Identification of binding partners using mass spectrometry

• Functional approaches:

  • Use of cytoskeleton-disrupting agents (cytochalasin D, latrunculin B, nocodazole)

  • Expression of dominant-negative forms of cytoskeletal regulators

  • siRNA knockdown of potential cytoskeletal interacting proteins

Since CEACAM4 contains an ITAM that becomes phosphorylated and recruits SH2 domain-containing proteins like Nck , which can link receptors to the actin cytoskeleton, focusing on actin dynamics during CEACAM4-mediated phagocytosis would be particularly informative.

How might high-throughput approaches be used to identify potential CEACAM4 functions?

Several high-throughput approaches could advance our understanding of CEACAM4 biology:

• Transcriptomic analysis:

  • RNA-seq of cells with manipulated CEACAM4 expression

  • Single-cell RNA-seq to identify CEACAM4-expressing cell populations

  • Comparison of transcriptional profiles before and after receptor engagement

• Proteomic approaches:

  • Phosphoproteomic analysis following CEACAM4 activation

  • Proximity labeling (BioID/TurboID) to identify the CEACAM4 interactome

  • Quantitative proteomics of phagosomes isolated from cells with active versus inactive CEACAM4

• CRISPR-Cas9 screening:

  • Genome-wide CRISPR screens to identify genes affecting CEACAM4-mediated phagocytosis

  • Focused screens targeting cytoskeletal regulators or signaling molecules

  • CRISPRi/CRISPRa screens to identify transcriptional regulators of CEACAM4 expression

• High-content imaging:

  • Automated microscopy to quantify CEACAM4-dependent phagocytosis under various conditions

  • Phenotypic profiling of cells with altered CEACAM4 signaling

These approaches could help identify unexpected functions and regulatory mechanisms for CEACAM4 beyond its established role in phagocytosis .

What is the potential role of CEACAM4 in human disease and immunity?

Based on its expression pattern and functional characteristics, CEACAM4 may play important roles in several aspects of human health and disease:

• Infectious disease:

  • Recognition and clearance of certain gram-negative bacteria

  • Potential target for bacterial evasion strategies

  • Primate-specific contribution to innate immunity

• Cancer biology:

  • Altered expression in certain malignancies (e.g., lung cancer)

  • Possible role in tumor cell recognition by phagocytes

  • Potential diagnostic or prognostic marker

• Inflammatory disorders:

  • Regulation of neutrophil function in inflammatory conditions

  • Possible involvement in autoimmune diseases where neutrophil function is dysregulated

• Therapeutic opportunities:

  • Targeting CEACAM4 to enhance bacterial clearance

  • Modulating CEACAM4 function to alter inflammatory responses

  • Using CEACAM4 antibodies for diagnostic purposes

Research in these areas would benefit from the development of improved tools, including monoclonal antibodies with different functional properties (neutralizing, activating) and recombinant proteins for in vitro and in vivo studies.

What validation steps are essential before using CEACAM4 antibodies in experimental procedures?

Before using CEACAM4 antibodies in research applications, comprehensive validation is necessary:

• Specificity testing:

  • Western blot analysis using positive control cell lines (HL-60, THP-1)

  • Direct ELISAs against all CEACAM family members to confirm lack of cross-reactivity

  • Testing on cells with CEACAM4 knockdown or knockout

  • Peptide competition assays

• Application-specific validation:

  • Optimization of antibody concentration for each application (Western blot: ~2 μg/mL)

  • Determination of optimal fixation and permeabilization methods for immunohistochemistry

  • Verification of recognition in both native and denatured forms

• Lot-to-lot consistency:

  • Testing new lots against previous lots for consistent results

  • Maintaining reference samples for comparison

• Positive and negative controls:

  • Include known CEACAM4-expressing cell lines as positive controls

  • Use cell lines lacking CEACAM4 expression as negative controls

  • Consider using cells transfected with CEACAM4 as additional controls

Thorough validation ensures reliable results and helps avoid misinterpretation of data due to antibody cross-reactivity, which is particularly important for CEACAM family research due to structural similarities between family members.

How can researchers optimize Western blot protocols for CEACAM4 detection?

Optimizing Western blot protocols for CEACAM4 detection requires attention to several key factors:

• Sample preparation:

  • Use appropriate lysis buffers containing phosphatase inhibitors to preserve phosphorylated forms

  • Include protease inhibitors to prevent degradation

  • Determine optimal protein loading (typically 20-50 μg total protein)

• Electrophoresis conditions:

  • CEACAM4 appears at approximately 30 kDa under reducing conditions

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Include molecular weight markers in the appropriate range

• Transfer and blocking:

  • PVDF membranes are suitable for CEACAM4 detection

  • Optimize transfer time and voltage for proteins in the 30 kDa range

  • Use 5% non-fat dry milk or 3-5% BSA in TBS-T for blocking

• Antibody incubation:

  • Primary antibody: Use anti-CEACAM4 at 2 μg/mL concentration

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: HRP-conjugated anti-mouse IgG antibody

  • Include proper washes between antibody incubations

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Consider more sensitive detection methods for low-abundance samples

  • Optimize exposure times to avoid saturation

These optimized conditions have been successfully used to detect CEACAM4 in various human cell lines including HL-60, THP-1, IMR-90, and NCI-H596, as well as in human lung cancer tissue .