CEACAM16 is a cell adhesion molecule primarily expressed in the tectorial membrane of the inner ear, where it stabilizes auditory function . Its dysregulation has been linked to hearing impairments . Beyond auditory roles, CEACAM16 is expressed in certain cancer types, including colorectal cancer, where it may serve as a diagnostic or therapeutic target .
The CEACAM16 Antibody, FITC conjugated, is optimized for:
Flow Cytometry: Detecting CEACAM16 expression in human cell populations .
Immunocytochemistry: Visualizing CEACAM16 in tissue or cell samples .
Optimal dilutions must be experimentally determined for each application .
For flow cytometry, pair with a compatible secondary antibody (e.g., PE-conjugated) .
CEACAM16 is essential for tectorial membrane integrity . Knockout models exhibit hearing loss, underscoring its role in mechanotransduction .
While not directly marketed for oncology, CEACAM16 is part of the broader CEACAM family (e.g., CEACAM1, CEACAM5) targeted in colorectal cancer diagnostics . A related antibody (6G5j) binds multiple CEACAMs, including CEACAM16, and has been tested for fluorescence-guided surgery .
| Antibody | Conjugation | Reactivity | Applications |
|---|---|---|---|
| CEACAM16 [SU-9D5] | Unconjugated | Human, Mouse | Flow Cytometry, IHC-P |
| CEACAM16 [2747B] | PE | Human | Flow Cytometry, ICC |
| CEACAM16 [FITC] | FITC | Human | Flow Cytometry, ICC |
Key Difference: The FITC-conjugated variant is primarily used for fluorescence-based assays, while unconjugated versions (e.g., [SU-9D5]) are versatile for immunohistochemistry .
CEACAM16 is a member of the carcinoembryonic antigen family of adhesion proteins expressed in mammalian outer hair cells. It localizes to the tips of the tallest stereocilia and the tectorial membrane in the inner ear. This specific localization suggests CEACAM16 plays a crucial role in maintaining the integrity of the tectorial membrane and the connection between outer hair cell stereocilia and the tectorial membrane, which is essential for mechanical amplification in the hearing process. Mutations in the CEACAM16 gene are associated with autosomal dominant nonsyndromic hearing loss (ADNSHL), specifically at the DFNA4 locus, emphasizing its significance in hearing research .
CEACAM16 antibodies are utilized in multiple research applications including:
Western Blotting (WB) for protein detection and quantification
Immunofluorescence on both cultured cells (IF-cc) and paraffin-embedded sections (IF-p)
Immunohistochemistry (IHC) for localizing the protein in tissue sections
Flow cytometry for detecting expression in specific cell populations
ELISA for quantitative protein detection in biological samples
These applications enable researchers to investigate CEACAM16 expression, localization, and function in various experimental contexts, particularly in inner ear and hearing research.
FITC (Fluorescein isothiocyanate) conjugation of CEACAM16 antibodies provides several methodological advantages for researchers:
Direct detection without secondary antibodies, reducing background and non-specific binding
Enables multiplexing with other fluorophore-conjugated antibodies for co-localization studies
Compatible with standard fluorescence microscopy equipment using blue excitation (488 nm)
Suitable for flow cytometry applications with minimal spectral overlap when used with appropriate filter sets
Allows for real-time visualization in certain applications
When working with FITC-conjugated antibodies, researchers should be mindful of potential photobleaching and should store antibodies protected from light to maintain fluorescence intensity .
CEACAM16 antibodies demonstrate varied species reactivity profiles depending on the specific antibody clone and epitope. Based on available data, many CEACAM16 antibodies show reactivity with:
Human (primary research target)
Mouse (common model organism)
Rat (research model)
Other mammals including cow, pig, guinea pig, dog, horse, and rabbit
Comprehensive validation of FITC-conjugated CEACAM16 antibodies should include:
Positive and negative control tissues: Compare inner ear tissues (positive) with tissues known not to express CEACAM16 such as peripheral blood monocytes or lymphocytes
Competitive inhibition assays: Pre-incubate antibody with excess purified CEACAM16 protein to block specific binding
Knockout/knockdown validation: Test antibody in CEACAM16 knockout models or siRNA-treated cells
Correlation with mRNA expression: Verify protein detection correlates with RT-qPCR results
Multiple antibody validation: Compare staining patterns with antibodies targeting different CEACAM16 epitopes
Western blot confirmation: Verify specificity by detection of the expected ~45.9 kDa band (may appear at ~53 kDa with glycosylation)
These validation steps are essential to avoid misinterpretation of results in complex experimental systems, particularly when investigating subtle changes in protein expression or localization.
For optimal immunofluorescence results with FITC-conjugated CEACAM16 antibodies:
Fixation: 4% paraformaldehyde fixation for 15-20 minutes preserves CEACAM16 structure while maintaining fluorescent signal
Permeabilization: Gentle permeabilization with 0.1-0.3% Triton X-100 is recommended for intracellular detection
Blocking: Use 5-10% normal serum from the same species as the secondary antibody with 1% BSA to reduce background
Antibody concentration: Optimize antibody dilution (typically 1:50 to 1:500) based on signal-to-noise ratio
Incubation conditions: Overnight incubation at 4°C typically yields the best signal-to-noise ratio
Counterstaining: DAPI nuclear staining provides context without spectral overlap with FITC
Mounting: Use anti-fade mounting medium to minimize photobleaching
Controls: Include isotype controls at equivalent concentration and secondary-only controls
Based on published results, CEACAM16 proteins are primarily distributed in the cell cytoplasm without regional aggregation, consistent with its nature as a secreted protein .
Mutations in CEACAM16, particularly those associated with hearing loss, may impact antibody-based detection in several ways:
Epitope masking: Conformational changes may hide antibody binding sites
Altered expression levels: The p.Arg255Gly mutation has been shown to increase CEACAM16 protein expression compared to wild-type
Subcellular localization changes: While no differences in localization were observed with the p.Arg255Gly mutation, other mutations might affect protein trafficking
Post-translational modification differences: Mutations may alter glycosylation patterns, affecting apparent molecular weight
Protein stability changes: Some mutations might affect protein half-life, altering steady-state levels
Research indicates that certain mutations like p.Arg255Gly can significantly increase both intracellular and secreted CEACAM16 protein levels, which would be detectable as increased fluorescence intensity when using FITC-conjugated antibodies. This highlights the importance of quantitative analysis in immunofluorescence studies of CEACAM16 variants .
For accurate quantification of CEACAM16 using FITC-conjugated antibodies:
Flow cytometry:
Single-cell quantification of CEACAM16 expression
Use appropriate compensation controls for multicolor panels
Include quantification beads for standardization
Quantitative fluorescence microscopy:
Maintain consistent exposure settings between samples
Use internal reference standards
Apply background subtraction and thresholding consistently
Analyze multiple fields/regions for representative results
Microplate-based fluorescence:
Develop solid-phase assays using capture antibodies
Create standard curves with recombinant CEACAM16
Account for autofluorescence in tissue/cell samples
Western blot with in-gel fluorescence detection:
Run samples alongside concentration standards
Use software for densitometric analysis
Normalize to appropriate loading controls
Research demonstrates that ELISA-based methods can effectively detect differences in CEACAM16 levels between wild-type and mutant proteins. The p.Arg255Gly mutation showed significantly higher protein levels compared to wild-type CEACAM16 (p < 0.01), indicating the sensitivity of quantitative immunoassays for this protein .
CEACAM16 has been shown to interact with α-tectorin, a key structural component of the tectorial membrane. This interaction has significant implications for experimental design:
Co-immunoprecipitation studies confirm physical interaction between CEACAM16 and α-tectorin
Immunofluorescence reveals co-localization of these proteins in the tectorial membrane
The interaction appears essential for maintaining structural integrity of the tectorial membrane
Disruption of this interaction may underlie the pathology of CEACAM16-associated hearing loss
When designing experiments to study CEACAM16, researchers should consider this interaction and how experimental conditions might affect protein-protein interactions. Co-immunoprecipitation followed by Western blotting can confirm these interactions, while proximity ligation assays can visualize them in situ .
Detecting CEACAM16 in cochlear tissues presents unique challenges requiring specialized approaches:
Tissue processing:
Careful decalcification procedures must preserve protein epitopes
Cryosectioning often preserves antigenicity better than paraffin embedding
Section thickness (8-12 μm) is critical for good signal penetration
Fixation considerations:
Standard formaldehyde fixation may mask CEACAM16 epitopes
Acetone or methanol fixation may better preserve certain epitopes
Antigen retrieval methods should be optimized specifically for inner ear tissues
Detection sensitivity:
Signal amplification techniques (TSA) may be necessary for low abundance detection
FITC-conjugated primary antibodies provide direct detection with reduced background
Consider confocal microscopy for precise localization studies
Anatomical reference:
Co-staining with structural markers helps identify specific cochlear regions
Include developmental time-points as CEACAM16 expression is temporally regulated
When analyzing cochlear tissues, researchers should be aware that CEACAM16 localizes specifically to the tips of the tallest stereocilia and throughout the tectorial membrane, requiring high-resolution imaging techniques for accurate detection and localization .
Distinguishing normal and mutant CEACAM16 requires specialized experimental approaches:
Epitope-specific antibodies:
Antibodies targeting mutation-specific epitopes can directly distinguish variants
For common mutations like p.Arg255Gly, custom antibodies may be developed
Functional assays:
Secretion assays comparing wild-type and mutant protein levels
Western blot analysis showing altered mobility or expression levels
Immunofluorescence revealing differences in localization patterns
Expression analysis:
RT-qPCR to quantify mRNA expression differences
ELISA to measure protein concentration differences in culture media
Flow cytometry to assess cellular protein level differences
Protein-protein interaction studies:
Co-immunoprecipitation to assess altered binding to α-tectorin
Surface plasmon resonance to measure binding kinetic differences
Research has demonstrated that the p.Arg255Gly mutation results in significantly higher protein expression both intracellularly and in secreted form compared to wild-type CEACAM16. This mutation does not appear to alter subcellular localization but does increase protein levels, which can be detected and quantified using antibody-based methods .
For successful multiplexing of FITC-conjugated CEACAM16 antibodies with other fluorophores:
Spectral considerations:
FITC emission (peak ~520 nm) overlaps with PE, thus careful compensation is required
Optimal partners include APC (far-red) and Pacific Blue/BV421 (violet) fluorophores
Avoid tandem dyes that include FITC in their energy transfer pathway
Sequential staining approach:
Apply FITC-conjugated primary antibodies first, followed by other direct conjugates
For indirect detection of other targets, block remaining FITC antibody binding sites
Microscopy settings:
Use narrow bandpass filters to minimize spectral overlap
Sequential scanning in confocal microscopy reduces bleed-through
Consider spectral unmixing for closely overlapping fluorophores
Controls for multiplexing:
Single-stained controls for compensation calculation
Fluorescence-minus-one (FMO) controls to set gating boundaries
Isotype-matched controls for each fluorophore
For studying CEACAM16 in relation to other tectorial membrane proteins, researchers have successfully employed multiplexed approaches combining FITC-conjugated antibodies with longer-wavelength fluorophores to minimize spectral overlap while enabling co-localization analysis .
| Challenge | Possible Causes | Solutions |
|---|---|---|
| Weak signal | Low expression of target protein, photobleaching, suboptimal antibody concentration | Increase antibody concentration, use anti-fade mounting media, employ signal amplification techniques, optimize fixation methods |
| High background | Non-specific binding, autofluorescence, insufficient blocking | Increase blocking time/concentration, add 0.1-0.3% Triton X-100 to blocking buffer, include species-matched serum, use Sudan Black B to reduce tissue autofluorescence |
| Non-specific binding | Cross-reactivity with related CEACAM family proteins | Pre-adsorb antibody with related proteins, validate with knockout controls, use monoclonal antibodies with confirmed specificity |
| Inconsistent results | Lot-to-lot antibody variation, inconsistent sample preparation | Use the same antibody lot for comparable experiments, standardize all protocols, include positive control samples |
| Signal variability in tissue sections | Regional differences in protein expression, fixation artifacts | Use consistent anatomical landmarks, standardize fixation times, process control and experimental samples simultaneously |
When investigating CEACAM16 in cochlear tissues, researchers should be particularly attentive to fixation methods, as over-fixation can mask epitopes while under-fixation may compromise tissue morphology .
Optimize protein extraction using buffers containing 1% NP-40 or Triton X-100
Include protease inhibitors to prevent degradation
Use 7.5-10% gels for optimal resolution of the ~45-53 kDa CEACAM16 protein
Transfer at lower voltage overnight for complete transfer of glycosylated proteins
Block with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature
Test multiple fixatives (4% PFA, methanol, or acetone) to determine optimal epitope preservation
For inner ear tissues, extend permeabilization time to ensure antibody penetration
Include 0.1% BSA in wash buffers to reduce non-specific binding
Incubate with primary antibody at 4°C overnight for best signal-to-noise ratio
Mount with anti-fade medium containing DAPI for nuclear counterstaining
Optimize cell permeabilization if detecting intracellular CEACAM16
Include viability dye to exclude dead cells, which can bind antibodies non-specifically
Titrate antibody concentration using positive control samples
Include 2% normal mouse serum to block Fc receptors
Set PMT voltages using unstained and single-stained controls
These application-specific optimizations enhance detection sensitivity while minimizing background and non-specific binding, crucial for studying CEACAM16 in complex biological systems .
For investigating CEACAM16 dynamics and protein-protein interactions, researchers can employ:
Fluorescence Recovery After Photobleaching (FRAP):
Monitors protein mobility in live cells expressing CEACAM16-GFP fusion proteins
FITC-antibody fragments can be used for pulse-chase studies in certain systems
Reveals secretion dynamics and potential membrane associations
Förster Resonance Energy Transfer (FRET):
Pair FITC-conjugated CEACAM16 antibodies with acceptor fluorophore-labeled α-tectorin antibodies
Detects direct protein-protein interactions within 10 nm
Useful for confirming structural associations in the tectorial membrane
Proximity Ligation Assay (PLA):
Detects protein interactions with greater sensitivity than conventional co-localization
Can be used to map CEACAM16 interaction networks in cochlear tissues
Amplifies signal only when target proteins are within 40 nm proximity
Super-resolution microscopy techniques:
STED, STORM, or PALM imaging overcomes diffraction limit for nanoscale localization
Reveals precise distribution of CEACAM16 within stereocilia and tectorial membrane
Required for detailed co-localization with structural proteins at molecular scale
These advanced techniques provide deeper insights into CEACAM16 function beyond simple localization, helping elucidate the molecular mechanisms of its role in hearing and the pathophysiology of associated hearing loss .
Differentiating CEACAM16 from other family members requires careful consideration of:
Antibody epitope selection:
Target unique regions (amino acids 201-300 or 323-414) that differ from other CEACAM proteins
Avoid antibodies targeting conserved domains shared across the family
Validate specificity using recombinant proteins representing multiple family members
Expression pattern analysis:
CEACAM16 is predominantly expressed in cochlear outer hair cells
Unlike CEACAM8 (CD66b), CEACAM16 is not expressed by peripheral blood granulocytes
Tissue-specific expression provides a control for antibody specificity
Molecular weight discrimination:
Human CEACAM16 has a theoretical mass of 45.9 kDa, appearing at ~53 kDa with glycosylation
Compare with known molecular weights of other CEACAM proteins (CEACAM8: 95-100 kDa)
Use gradient gels for optimal separation of different family members
Cross-validation approaches:
Confirm results with multiple antibodies targeting different CEACAM16 epitopes
Correlate protein detection with mRNA expression by RT-qPCR using gene-specific primers
Use gene silencing or knockout models to verify antibody specificity
These strategies are essential when studying CEACAM proteins in complex systems where multiple family members may be expressed, ensuring specific detection of CEACAM16 without cross-reactivity artifacts .
FITC-conjugated CEACAM16 antibodies offer valuable applications for hearing loss research:
Genotype-phenotype correlation studies:
Compare CEACAM16 expression and localization across various mutation types
Correlate protein expression patterns with audiometric findings
Track age-related changes in protein expression in models of progressive hearing loss
Therapeutic development screening:
Evaluate compounds that might stabilize mutant CEACAM16 structure
Screen for agents that modulate protein-protein interactions with α-tectorin
Assess gene therapy approaches targeting CEACAM16 expression
Diagnostic applications:
Develop molecular phenotyping of cochlear explants from patients
Create screening assays for CEACAM16 functionality
Establish biomarkers for early detection of ADNSHL
Developmental studies:
Map temporal expression during inner ear development
Identify critical periods for CEACAM16 function in hearing development
Investigate regenerative approaches for hearing restoration
These research applications build on findings that mutations in CEACAM16 are associated with autosomal dominant nonsyndromic hearing loss (DFNA4B) and autosomal recessive hearing loss (DFNB113), highlighting the protein's critical role in normal hearing function .
Emerging techniques for quantitative analysis of CEACAM16 include:
Quantitative image cytometry:
High-content screening platforms for automated analysis of fluorescence intensity
Machine learning algorithms for pattern recognition in complex tissues
Tissue cytometry for single-cell quantification within intact cochlear sections
Spatial transcriptomics integration:
Correlating protein detection with mRNA expression in situ
Combined fluorescence in situ hybridization (FISH) with immunofluorescence
Spatial resolution of CEACAM16 expression in relation to functional hearing zones
Digital pathology approaches:
Whole-slide imaging with automated quantification
Multi-spectral analysis for distinguishing FITC signal from autofluorescence
3D reconstruction of protein distribution through confocal z-stacks
Nanoscale quantification methods:
Single-molecule detection using quantum dots
DNA-PAINT for quantitative super-resolution imaging
Correlative light and electron microscopy for ultrastructural context
These technologies enable more precise quantification of CEACAM16 expression changes in experimental models and patient samples, facilitating mechanistic studies of hearing loss progression and potential therapeutic interventions .
| Experimental Model | Advantages | Limitations | Optimal Applications |
|---|---|---|---|
| Mouse knockout models | Complete gene deletion, well-characterized auditory system, genetic manipulation options | Species differences in hearing range, cochlear access challenges | Phenotype characterization, development studies, in vivo function |
| Cochlear explant cultures | Maintains tissue architecture, allows manipulation and live imaging, accessible for antibody application | Short-term viability, technical challenges in preparation | Protein localization, trafficking studies, acute interventions |
| Cell line transfection systems (HEK293T) | Easy genetic manipulation, high transfection efficiency, biochemical assays | Non-native cellular environment, lacks cochlear context | Protein-protein interactions, mutation analysis, secretion studies |
| Inner ear organoids | 3D structure, human cell options, developmental processes | Incomplete maturation, variability between preparations | Developmental studies, patient-specific disease modeling |
| Zebrafish lateral line | Optical transparency, rapid development, genetic manipulation | Evolutionary distance from mammals | High-throughput screening, live imaging of hair cell function |
Research has demonstrated that HEK293T cells transfected with wild-type or mutant CEACAM16 provide a valuable system for investigating protein secretion, localization, and expression levels. This system has successfully revealed that the p.Arg255Gly mutation increases both intracellular and secreted CEACAM16 levels compared to wild-type, providing insights into potential disease mechanisms .
CEACAM16 antibodies could advance therapeutic approaches for hearing loss through:
Targeted drug delivery systems:
Antibody-drug conjugates specifically targeting the tectorial membrane
Nanoparticle carriers functionalized with CEACAM16-targeting fragments
Development of bispecific antibodies targeting both CEACAM16 and therapeutic targets
Diagnostic companion applications:
Patient stratification for clinical trials based on CEACAM16 expression patterns
Monitoring treatment efficacy through protein expression changes
Early detection of tectorial membrane integrity compromise
Therapeutic screening platforms:
High-content screening using FITC-conjugated antibodies to assess compound effects
Functional recovery assessment in ex vivo cochlear models
Evaluation of gene therapy approaches targeting CEACAM16 expression
Precision medicine approaches:
Antibody-based assays to determine mutation-specific therapeutic strategies
Monitoring of protein misfolding correction therapies
Assessment of protein replacement therapy efficacy
While therapeutic applications remain experimental, the fundamental understanding of CEACAM16's role in hearing provides a foundation for developing targeted interventions for CEACAM16-associated hearing loss, particularly for autosomal dominant nonsyndromic deafness at the DFNA4 locus and autosomal recessive hearing loss at the DFNB113 locus .