CEACAM5 Antibody, Biotin conjugated

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

CEACAM5 (Carcinoembryonic Antigen-related Cell Adhesion Molecule 5), also known as CD66e or CEA, is a membrane-bound glycoprotein with a molecular weight ranging from 80-200 kDa depending on glycosylation patterns. The canonical human CEACAM5 protein consists of 702 amino acid residues with a base mass of 76.8 kDa before post-translational modifications. It functions primarily as a cell adhesion molecule and plays roles in the apoptotic pathway .

What are the main applications for biotin-conjugated CEACAM5 antibodies in research?

Biotin-conjugated CEACAM5 antibodies are versatile tools in research settings with multiple established applications:

The biotin conjugation provides significant advantages over unconjugated antibodies, including signal amplification through the strong biotin-streptavidin interaction, flexible detection options, and compatibility with multi-parameter assays. These applications are particularly valuable in cancer research for detecting circulating tumor cells and in virology studies examining CEACAM5's role in viral attachment .

How does the C66/1292 clone compare to other CEACAM5 antibody clones?

The C66/1292 clone exhibits several distinctive characteristics compared to other commercially available CEACAM5 antibody clones:

• Specificity profile: The C66/1292 clone recognizes different members of the CEA family (80-200kDa) but importantly does not cross-react with nonspecific cross-reacting antigen (NCA) or human polymorphonuclear leukocytes .

• Epitope recognition: This clone is raised against recombinant full-length human CEACAM5/CD66e protein (UniProt: P06731), allowing for recognition of the native conformation of the protein .

• Tissue reactivity: C66/1292 shows no reaction with a variety of normal tissues but exhibits strong reactivity with adenocarcinomas from various origins (lung, colon, stomach, esophagus, pancreas, gallbladder, urachus, salivary gland, ovary, and endocervix) .

• Application versatility: While many clones are restricted to certain applications, C66/1292 has demonstrated effectiveness across multiple techniques including CyTOF, flow cytometry, and immunocytochemistry/immunofluorescence .

When selecting between clones, researchers should consider these differences in relation to their specific experimental requirements, particularly regarding cross-reactivity concerns and intended applications.

How can I optimize biotin-conjugated CEACAM5 antibody staining for dual-parameter flow cytometry?

Optimizing biotin-conjugated CEACAM5 antibody staining for dual-parameter flow cytometry requires careful consideration of several technical factors:

• Titration optimization: Perform a comprehensive antibody titration series (typically 0.1-10 μg/mL) to determine the optimal signal-to-noise ratio. Monitor both positive signal intensity and background staining across concentrations. The optimal concentration is often lower than manufacturer-suggested starting concentrations due to the signal amplification provided by the biotin-streptavidin system .

• Sequential staining protocol:

  • Fix cells in 2% paraformaldehyde for 20 minutes at room temperature

  • Permeabilize (if detecting intracellular epitopes) with 0.1% saponin or 0.1% Triton X-100

  • Block with 2% serum matching secondary reagent species for 30 minutes

  • Incubate with primary biotin-conjugated CEACAM5 antibody for 45-60 minutes

  • Wash three times with PBS containing 0.5% BSA

  • Incubate with fluorochrome-conjugated streptavidin for 30 minutes

  • Add additional directly conjugated antibodies for multiparameter analysis

  • Wash three times before analysis

• Streptavidin selection: Choose streptavidin conjugates in channels with minimal spectral overlap with other markers in your panel. For CEACAM5, APC or PE-Cy7 streptavidin conjugates often provide good separation between positive and negative populations .

• Control strategy: Include cells known to be CEACAM5 positive (such as certain adenocarcinoma cell lines) and confirmed negative cells. Additionally, include FMO (fluorescence minus one) controls and isotype-biotin controls conjugated to the same streptavidin fluorophore .

What are the critical validation steps when using biotin-conjugated CEACAM5 antibodies for detecting viral attachment?

When investigating CEACAM5's role as an attachment factor for viruses such as MERS-CoV, rigorous validation steps are essential to ensure reliable results:

• Confirmation of antibody blocking capacity:

  • Pre-incubate target cells with serial dilutions of the biotin-conjugated CEACAM5 antibody

  • Assess inhibition of viral attachment using virus overlay protein binding assay (VOPBA)

  • Confirm with complementary techniques such as flow cytometry to measure viral protein binding

• Specificity controls:

  • Include parallel experiments with isotype-matched biotin-conjugated control antibodies

  • Perform knockdown/knockout validation using CEACAM5 siRNA or CRISPR-engineered cell lines

  • Complement binding studies with co-immunoprecipitation to verify direct interaction

• Cross-validation with unconjugated antibodies:

  • Compare results between biotin-conjugated and unconjugated versions of the same clone

  • Test whether biotin conjugation affects epitope recognition or blocking functionality

• Viral attachment quantification:

  • Develop a standardized quantification protocol using fluorescently labeled viral particles

  • Establish dose-response relationships between antibody concentration and viral attachment inhibition

  • Calculate IC50 values to enable comparison between experimental conditions

Research has demonstrated that CEACAM5 functions as a novel attachment factor facilitating MERS-CoV entry, making this validation approach particularly relevant for coronavirus research and potential therapeutic development .

How should I address potential cross-reactivity with other CEACAM family members?

The CEACAM family contains multiple homologous members that can complicate experimental interpretation due to potential cross-reactivity. When working with biotin-conjugated CEACAM5 antibodies, implement these strategies to address this challenge:

• Comprehensive selectivity testing:

  • Express individual recombinant CEACAM family members (CEACAM1, CEACAM3, CEACAM6, CEACAM7, CEACAM8) in a null cell line

  • Perform parallel immunodetection to quantify relative binding affinities

  • Present data as normalized binding ratios between target and potential cross-reactive proteins

• Epitope mapping:

  • Utilize deletion mutants or peptide arrays to identify the specific epitope recognized by the CEACAM5 antibody

  • Select antibodies targeting regions with lower sequence homology between family members

  • The C66/1292 clone has documented specificity for CEACAM5 without cross-reactivity to nonspecific cross-reacting antigen (NCA)

• Complementary validation approaches:

  • Combine antibody-based detection with mass spectrometry identification

  • Implement genetic targeting via CRISPR or siRNA for each CEACAM family member

  • Utilize RT-qPCR to correlate protein detection with transcript expression profiles

• Interpretation framework:

  • Document known cross-reactivities in experimental designs

  • Include appropriate positive controls for each potential cross-reactive family member

  • Consider using antibody cocktails recognizing distinct epitopes to increase specificity

This methodical approach is particularly important when investigating CEACAM5 in tissues known to express multiple family members, such as intestinal epithelium or certain cancer types .

How can biotin-conjugated CEACAM5 antibodies be used to study tumor microenvironments?

Biotin-conjugated CEACAM5 antibodies offer several methodological advantages for investigating tumor microenvironments:

• Multiplex immunofluorescence profiling:

  • Combine biotin-conjugated CEACAM5 antibody with directly labeled antibodies against immune cell markers (CD8, CD4, CD68, etc.)

  • Utilize sequential streptavidin-fluorophore staining to amplify CEACAM5 signal

  • Employ multispectral imaging systems for co-expression analysis in spatial context

  • Quantify cellular relationships using nearest neighbor analysis or spatial correlation metrics

• Flow cytometry-based characterization:

  • Process tumor samples into single-cell suspensions

  • Stain with biotin-conjugated CEACAM5 and immune cell markers

  • Analyze co-expression patterns to identify cell-cell interactions

  • Sort CEACAM5+ and CEACAM5- populations for downstream functional assays

• CyTOF (mass cytometry) applications:

  • Include biotin-conjugated CEACAM5 in metal-tagged antibody panels

  • Perform high-dimensional analysis (30+ parameters)

  • Apply algorithms like UMAP or t-SNE for population identification

  • Correlate CEACAM5 expression with functional markers and cell states

CEACAM5 expression patterns in tumors reveal important biological insights, as this protein is synthesized during fetal development, silenced in most adult tissues, and then re-expressed in carcinomas, particularly adenocarcinomas from various origins. This differential expression makes it a valuable marker for understanding tumor heterogeneity and progression .

What methods can optimize detection of CEACAM5 in virus-host interaction studies?

When investigating CEACAM5's role in virus-host interactions, particularly as an attachment factor for MERS-CoV, specialized methodological approaches can enhance detection sensitivity and specificity:

• Synchronized infection protocols:

  • Pre-cool cells to 4°C to inhibit endocytosis

  • Add viral particles and allow binding for 1 hour at 4°C

  • Fix cells without permeabilization to preserve surface-bound virus

  • Stain with biotin-conjugated CEACAM5 antibody and virus-specific antibodies

  • Analyze co-localization using confocal microscopy or super-resolution techniques

• Real-time virus-receptor tracking:

  • Transfect cells with CEACAM5-fluorescent protein fusion constructs

  • Label viral particles with lipophilic dyes or tagged capsid proteins

  • Perform live-cell imaging using spinning disk confocal microscopy

  • Quantify co-localization coefficients over time during viral attachment

• Proximity ligation assay optimization:

  • Use biotin-conjugated CEACAM5 antibody with streptavidin-oligonucleotide conjugates

  • Pair with virus-specific antibodies conjugated to complementary oligonucleotides

  • Amplify signal only when CEACAM5 and viral proteins are in close proximity (<40 nm)

  • Quantify interaction events at single-molecule resolution

• FRET-based binding assays:

  • Develop a system using biotin-streptavidin-fluorophore complexes as FRET donors

  • Use fluorescently tagged viral proteins as FRET acceptors

  • Measure energy transfer as evidence of molecular proximity

  • Calculate binding kinetics based on FRET efficiency changes

These methodologies have proven valuable in establishing CEACAM5 as a novel attachment factor that facilitates MERS-CoV entry, highlighting potential new targets for antiviral strategy development .

How can I differentiate between membrane-bound and soluble CEACAM5 forms in research samples?

Distinguishing between membrane-bound and soluble forms of CEACAM5 requires specialized analytical approaches:

• Differential centrifugation protocol:

  • Collect biological samples (cell culture supernatant, serum, etc.)

  • Perform sequential centrifugation: 300g (cells), 3,000g (debris), 10,000g (large vesicles), 100,000g (exosomes and membrane fragments)

  • Analyze each fraction using biotin-conjugated CEACAM5 antibodies

  • Compare distribution patterns across fractions

• Flow cytometry discrimination strategy:

  • For membrane-bound detection: Stain intact cells with biotin-conjugated CEACAM5 antibody

  • For soluble form analysis: Use bead-based capture systems with anti-CEACAM5 coating

  • Apply the same biotin-conjugated CEACAM5 antibody as detection reagent

  • Calibrate with recombinant standards to enable quantification

• Biochemical separation approach:

  • Perform size exclusion chromatography to separate membrane fragments from soluble proteins

  • Analyze fractions with a sandwich ELISA using biotin-conjugated CEACAM5 antibody

  • Confirm membrane association through detergent phase separation experiments

  • Validate findings with Western blot analysis of fractions

• Microscopy-based distinction:

  Parameter	Membrane-bound CEACAM5	Soluble CEACAM5
  Localization	Cell periphery pattern	Diffuse staining
  Co-localization	With membrane markers	No specific pattern
  Internalization	Dynamic endocytosis	Static distribution
  Pattern after fixation	Sharp boundary staining	Background-like signal

These methodological approaches are important because CEACAM5 exists in both forms: the canonical membrane-bound form (76.8 kDa core protein, 80-200 kDa with glycosylation) that functions in cell adhesion, and soluble forms that may be released through proteolytic cleavage or alternative splicing .

What are the optimal storage and handling conditions for maintaining biotin-conjugated CEACAM5 antibody activity?

Maintaining the functional integrity of biotin-conjugated CEACAM5 antibodies requires strict adherence to specialized storage and handling protocols:

• Storage temperature requirements:

  • Store at 4°C in the dark for short-term (1-2 months)

  • For long-term stability, aliquot and store at -20°C (avoid repeated freeze-thaw cycles)

  • Never store biotin-conjugated antibodies at room temperature due to accelerated degradation

  • Monitor temperature fluctuations with logging devices in critical storage units

• Buffer composition considerations:

  • Maintain in PBS (pH 7.2-7.4) with 0.05% sodium azide as preservative

  • Avoid buffers containing primary amines (Tris) which can interfere with biotin activity

  • For enhanced stability, supplementation with 1% BSA or 50% glycerol is recommended

  • Consider oxygen-scavenging additives to prevent oxidative damage to biotin

• Aliquoting strategy:

  • Prepare single-use aliquots in volumes appropriate for typical experiments

  • Use sterile, low-protein binding microcentrifuge tubes

  • Include date of aliquoting and recommended expiration dates on labels

  • Maintain detailed logs of freeze-thaw cycles for each antibody lot

• Quality control procedures:

  • Implement regular validation testing of stored antibodies

  • Include positive controls from previous experiments to verify consistent activity

  • Monitor biotin conjugation stability using streptavidin binding assays

  • Document signal intensity metrics over time to detect potential degradation

Additional precautions specific to biotin conjugates include protection from direct light exposure, which can accelerate photodegradation of both the biotin moiety and fluorescent streptavidin conjugates used for detection .

What controls are essential when using biotin-conjugated CEACAM5 antibodies in immunoassays?

A comprehensive control strategy is critical for ensuring reliable results when working with biotin-conjugated CEACAM5 antibodies:

• Antibody specificity controls:

  • Positive tissue/cell controls: Adenocarcinoma samples known to express CEACAM5

  • Negative tissue/cell controls: Normal tissues with documented absence of CEACAM5 expression

  • Blocking controls: Pre-incubation with recombinant CEACAM5 protein to confirm specificity

  • Genetic controls: CEACAM5 knockout/knockdown cells compared to wild-type

• Conjugation-specific controls:

  • Unconjugated primary antibody control: Same clone without biotin to assess conjugation effects

  • Biotin blocking control: Pre-treatment with free biotin or streptavidin to test for endogenous biotin

  • Secondary-only control: Streptavidin-conjugate alone to detect endogenous biotin

  • Isotype-biotin control: Biotin-conjugated non-specific antibody of the same isotype and concentration

• Signal validation controls:

  • Titration series: Serial dilutions to confirm proportional signal reduction

  • Signal amplification controls: Direct comparison between detection methods

  • Cross-platform validation: Confirmation of findings using independent techniques

• Technical controls:

  Control Type	Purpose	Implementation
  Fluorescence compensation	Correct spectral overlap	Single-stained controls for each fluorophore
  Instrument calibration	Ensure consistent sensitivity	Standardized beads with known binding capacity
  Experimental replicate	Assess technical variation	Independent preparation of identical samples
  Lot-to-lot comparison	Monitor manufacturing consistency	Parallel testing of different antibody lots

This control framework addresses the core technical considerations when working with biotin-conjugated antibodies while accounting for the specific characteristics of CEACAM5 expression, which is predominantly found in adenocarcinomas but absent in normal tissues, benign glands, stroma, and malignant prostatic cells .

How can I troubleshoot weak or non-specific staining with biotin-conjugated CEACAM5 antibodies?

When encountering staining issues with biotin-conjugated CEACAM5 antibodies, implement this systematic troubleshooting approach:

• Weak or absent signal problems:

  • Epitope retrieval optimization: For formalin-fixed tissues, test different retrieval methods (heat-induced vs. enzymatic) and pH conditions (pH 6.0 citrate vs. pH 9.0 EDTA)

  • Incubation parameters: Extend primary antibody incubation time (overnight at 4°C) and increase concentration incrementally

  • Amplification strategies: Implement tyramide signal amplification or multi-layer streptavidin systems

  • Antigen accessibility: Evaluate different permeabilization protocols (0.1-0.5% Triton X-100, 0.05-0.5% saponin, or methanol treatment)

• High background or non-specific staining issues:

  • Blocking optimization: Test increased concentrations (5-10%) of serum or BSA blockers

  • Washing stringency: Implement additional wash steps with 0.1% Tween-20 or 0.05% Triton X-100

  • Endogenous biotin blocking: Pre-treat samples with avidin-biotin blocking kit

  • Endogenous peroxidase quenching: If using HRP detection, treat with 0.3% H₂O₂ in methanol for 30 minutes

• Tissue-specific optimization strategies:

  Tissue Type	Common Issue	Recommended Adjustment
  Colon carcinoma	High endogenous biotin	Avidin-biotin block + streptavidin-poly-HRP detection
  Lung adenocarcinoma	Autofluorescence	Sudan Black B treatment (0.1% in 70% ethanol)
  FFPE tissues	Epitope masking	Extended retrieval times (20-40 minutes)
  Frozen sections	Morphology loss	2% PFA post-fixation before staining

• Antibody performance assessment:

  • Validate with alternative detection methods (e.g., compare biotin-streptavidin to direct conjugates)

  • Test antibody on known positive controls (adenocarcinoma samples) to confirm functionality

  • If possible, compare multiple CEACAM5 antibody clones on identical samples

  • Consider potential interference from sample preparation methods (fixatives, embedding media)

These methodological solutions address the complex nature of CEACAM5 detection, particularly considering its variable expression levels across different adenocarcinoma types and its absence in normal tissues and benign lesions .

How can biotin-conjugated CEACAM5 antibodies be utilized in coronavirus research beyond MERS-CoV?

Recent discoveries about CEACAM5's role as a viral attachment factor open new research directions applicable to broader coronavirus research:

• Comparative coronavirus attachment studies:

  • Screen CEACAM5 binding across coronavirus families using biotin-conjugated antibodies as blocking agents

  • Implement virus overlay protein binding assays (VOPBA) with streptavidin-conjugated reporter systems

  • Develop competition assays between MERS-CoV and other coronaviruses for CEACAM5 binding

  • Correlate CEACAM5 expression with tropism patterns of different coronaviruses

• Therapeutic development platforms:

  • Utilize biotin-conjugated CEACAM5 antibodies to develop screening assays for attachment inhibitors

  • Create bifunctional molecules linking CEACAM5-binding domains to viral neutralizing fragments

  • Design decoy receptor approaches based on CEACAM5 binding sites

  • Establish humanized mouse models with CEACAM5 expression for in vivo validation

• Methodological protocol for variant analysis:

  • Clone spike proteins from coronavirus variants into pseudotyped virus systems

  • Pre-treat cells with titrated concentrations of biotin-conjugated CEACAM5 antibodies

  • Measure infection efficiency through reporter gene expression

  • Calculate IC50 values to quantify relative dependence on CEACAM5 across variants

• Structural biology applications:

  • Use biotin-conjugated antibodies for epitope mapping of CEACAM5-virus interaction sites

  • Employ proximity labeling techniques to identify molecular neighbors during viral attachment

  • Develop cryo-EM approaches utilizing biotin-streptavidin linkages as fiducial markers

  • Characterize conformational changes in CEACAM5 upon viral binding

These applications build upon the foundational discovery that CEACAM5 functions as a novel attachment factor facilitating MERS-CoV entry, potentially serving as a target for antiviral strategy development. Similar approaches could reveal whether CEACAM5 plays roles in other coronavirus infections .

What are the methodological considerations for using CEACAM5 antibodies in CyTOF (mass cytometry) experiments?

CyTOF (Cytometry by Time-of-Flight) presents unique opportunities and challenges when using biotin-conjugated CEACAM5 antibodies:

• Metal conjugation strategy options:

  • Direct conjugation: Replace biotin with metal tags using commercial conjugation kits

  • Streptavidin bridge: Use metal-tagged streptavidin to detect biotin-conjugated antibodies

  • Secondary detection: Apply metal-tagged anti-species antibodies against the primary antibody

• Panel design optimization for CEACAM5 detection:

  • Allocate high-abundance metals (e.g., 165Ho, 166Er) for potentially low-expression antigens like CEACAM5

  • Include appropriate isotype controls conjugated to the same metal

  • Design panels to include markers for potential CEACAM5-expressing cell populations

  • Consider redundant markers to confirm cell identities in high-dimensional space

• Sample preparation protocol modifications:

  Step	Standard Protocol	CEACAM5-Optimized Approach
  Fixation	1.6% PFA, 10 min	2% PFA, 20 min to preserve membrane structures
  Permeabilization	0.3% saponin	Gentler 0.1% saponin to maintain epitope accessibility
  Barcoding	Palladium-based	Verify compatibility with CEACAM5 epitope preservation
  Antibody incubation	30 min, RT	Extended to 45-60 min for optimal binding

• Data analysis considerations:

  • Implement supervised gating strategies focusing on known CEACAM5-positive populations

  • Apply dimensionality reduction techniques (t-SNE, UMAP) to identify novel CEACAM5+ populations

  • Perform clustering algorithms (PhenoGraph, FlowSOM) to categorize cell populations

  • Correlate CEACAM5 expression with functional and phenotypic markers across populations

These methodological adaptations account for CEACAM5's expression characteristics, including its presence on adenocarcinomas from various tissues and absence from normal tissues, enabling researchers to leverage the high-parameter capabilities of CyTOF for comprehensive tumor microenvironment analysis .

What are the future directions for CEACAM5 antibody applications in research?

The research landscape for biotin-conjugated CEACAM5 antibodies is evolving rapidly, with several promising directions:

• Integrated multi-omics approaches:

  • Combining antibody-based imaging with single-cell transcriptomics

  • Correlating CEACAM5 protein expression with genomic alterations in tumors

  • Developing spatial proteomics methods using biotin-conjugated CEACAM5 antibodies

  • Creating computational frameworks to integrate protein expression with metabolomic profiles

• Advanced therapeutic applications:

  • Antibody-drug conjugate development using CEACAM5 targeting

  • CAR-T cell engineering based on CEACAM5-binding domains

  • Bispecific antibodies linking CEACAM5 recognition with immune cell activation

  • Theranostic approaches combining imaging and therapeutic capabilities

• Viral pathogenesis research expansion:

  • Exploring CEACAM5's role in attachment mechanisms across viral families

  • Developing intervention strategies targeting CEACAM5-virus interactions

  • Creating animal models with humanized CEACAM5 for in vivo studies

  • Investigating evolutionary relationships between viral proteins and CEACAM5

• Technological innovations:

  • Nanobody and aptamer development against CEACAM5 epitopes

  • Proximity-based proteomics to map CEACAM5 interaction networks

  • Live-cell super-resolution imaging of CEACAM5 dynamics

  • AI-assisted antibody engineering for improved specificity and affinity