ICAM3 Antibody, Biotin conjugated

What is ICAM3 and why is it an important research target?

ICAM3 (Intercellular Adhesion Molecule 3), also known as CD50, is a 120-130 kDa type I membrane protein belonging to the immunoglobulin supergene family. It serves as a ligand for the leukocyte adhesion protein LFA-1 (integrin alpha-L/beta-2) and integrin alpha-D/beta-2 . ICAM3 plays crucial roles in immune cell interactions, particularly in the initial contact between dendritic cells and T cells that support primary immune responses . Additionally, ICAM3 contributes to apoptotic neutrophil phagocytosis by macrophages in association with integrin alpha-L/beta-2 . Its expression pattern on leukocytes, endothelial cells, and Langerhans cells—but notably absent on platelets and erythrocytes—makes it an important marker for immunological research .

What are the optimal storage conditions for biotin-conjugated ICAM3 antibodies?

Biotin-conjugated ICAM3 antibodies should be stored at 2-8°C and should not be frozen . Most commercial preparations are shipped at 4°C and supplied in Phosphate Buffered Saline (pH 7.4) with 15 mM Sodium Azide as a preservative . The recommended storage duration is typically up to 12 months from the date of receipt at -20 to -70°C as supplied, 1 month at 2-8°C under sterile conditions after reconstitution, or 6 months at -20 to -70°C under sterile conditions after reconstitution . To maintain antibody integrity, avoid repeated freeze-thaw cycles and protect from prolonged exposure to light, especially for detection systems relying on the biotin conjugation.

What are the primary applications for biotin-conjugated ICAM3 antibodies?

The primary validated applications for biotin-conjugated ICAM3 antibodies include:

For flow cytometry applications, biotin-conjugated antibodies offer flexibility as they can be detected using various streptavidin-conjugated fluorochromes, allowing researchers to design multi-color panels while avoiding fluorescence spectrum overlaps .

How does the MEM-171 clone compare to other ICAM3 antibody clones?

The MEM-171 clone specifically recognizes an extracellular epitope in the D2 domain of CD50 (ICAM-3) . This differs from other available clones such as:

• MEM-04: Binds to a different epitope and is suitable for FACS, IHC, and functional assays

• 2D11D1: Recognizes amino acids 30-203 and is validated for FACS and ELISA

• 2F8: Recognizes amino acids 46-197 and is validated for Western blot, ELISA, and IHC

The epitope specificity is crucial as it determines which molecular interactions might be blocked or detected. The MEM-171 clone's specificity for the D2 domain makes it particularly useful for studying ICAM3's role in cell-cell adhesion, as this domain contributes to integrin binding interactions .

What controls should be included when using biotin-conjugated ICAM3 antibodies?

For rigorous experimental design, include the following controls:

• Isotype Control: Use a biotin-conjugated mouse IgG1 isotype control (such as MOPC-21) at the same concentration to assess non-specific binding .

• Blocking Control: Pre-incubate cells with unconjugated ICAM3 antibody before adding the biotin-conjugated version to confirm epitope specificity.

• Positive Control: Include samples known to express ICAM3 (e.g., human peripheral blood mononuclear cells).

• Negative Control: Include samples lacking ICAM3 expression (e.g., platelets or erythrocytes) to confirm specificity .

• Streptavidin-Only Control: When using streptavidin detection systems, include a control without primary antibody to assess background from the detection reagent.

These controls help differentiate true positive signals from background, non-specific binding, or autofluorescence, significantly enhancing data reliability and interpretation accuracy.

How should sample preparation be optimized for ICAM3 detection with biotinylated antibodies?

Sample preparation should be optimized based on the specific application:

For flow cytometry:

• Use freshly isolated cells whenever possible

• Maintain cells at 4°C during staining to prevent internalization of surface molecules

• Use buffers containing sodium azide (0.05-0.1%) to inhibit endocytosis

• Include 1-2% protein (BSA or FBS) in staining buffer to reduce non-specific binding

• For multi-color panels, consider the biotin-streptavidin step carefully in your staining sequence

For immunocytochemistry:

• Fixation with 4% paraformaldehyde preserves ICAM3 epitopes better than methanol

• When studying ICAM3 on PBMCs, use protocols optimized for non-adherent cells

• Consider gentle permeabilization if studying internalized ICAM3

The biotin-streptavidin interaction is one of the strongest non-covalent biological interactions, providing excellent sensitivity, but optimization of blocking steps is crucial to minimize background, especially in tissues with endogenous biotin .

Advantages:

• Signal Amplification: The biotin-streptavidin system allows for signal amplification, as multiple streptavidin molecules can bind to a single biotinylated antibody.

• Flexibility: Researchers can select from various streptavidin conjugates (fluorochromes, enzymes) without needing different primary antibodies.

• Stability: Biotinylation typically affects antibody stability less than direct fluorochrome conjugation.

• Panel Design: Facilitates complex multi-color flow cytometry panels by allowing strategic use of fluorochromes.

Limitations:

• Additional Step: Requires a secondary detection step, increasing protocol complexity and time.

• Endogenous Biotin: Tissues with high endogenous biotin (liver, kidney) may produce background that requires blocking steps.

• Potential Cross-Reactivity: When using multiple biotinylated antibodies, sequential detection is necessary to prevent cross-reactivity.

• Reagent Penetration: In some tissue applications, the larger streptavidin complex may have reduced penetration compared to directly labeled antibodies.

The biotin-conjugated format is particularly valuable when signal amplification is needed or when experimental design requires flexibility in detection systems .

How can biotin-conjugated ICAM3 antibodies be utilized in studying the role of ICAM3 in dendritic cell-T cell interactions?

Biotin-conjugated ICAM3 antibodies provide valuable tools for investigating the critical role of ICAM3 in dendritic cell (DC)-T cell interactions:

• Co-localization Studies: Use biotin-ICAM3 antibodies with streptavidin-fluorochromes alongside DC-SIGN markers to visualize interaction sites using confocal microscopy.

• Blocking Experiments: Apply biotin-ICAM3 antibodies to selectively block ICAM3-DC-SIGN interactions without affecting other adhesion pathways, as the MEM-171 clone recognizes a specific extracellular epitope in the D2 domain .

• Flow Cytometry-Based Binding Assays: Quantify DC-T cell conjugate formation with and without ICAM3 blockade using the biotin-conjugated antibody.

• Immunoprecipitation of Protein Complexes: Use biotin-ICAM3 antibodies to pull down ICAM3 and associated proteins during different stages of DC-T cell interaction.

Research has shown that ICAM3 is a natural ligand of DC-SIGN on DCs and is highly expressed on T cell surfaces. DC-SIGN binds ICAM3 with high affinity, making this interaction crucial for the initial contact between DCs and T cells that supports primary immune responses . Studies comparing ICAM3-Fc fusion proteins with antibody approaches have demonstrated that despite stronger affinity of certain antibodies for DC-SIGN, ICAM3-Fc induced more efficient cross-presentation in some contexts .

What methodologies enable the use of biotin-ICAM3 antibodies in targeted nanoparticle delivery systems?

Biotin-conjugated ICAM3 antibodies can be integrated into nanoparticle delivery systems through several methodological approaches:

• Streptavidin-Coated Nanoparticles: Utilize pre-formed streptavidin-coated nanoparticles that can directly capture biotinylated ICAM3 antibodies with high affinity.

• Two-Step Conjugation Process: First, biotinylated carrier systems (like PLGA nanoparticles) can be prepared, followed by addition of biotin-ICAM3 antibodies and streptavidin as a crosslinker .

This approach has been explored for targeted vaccine delivery, where PLGA nanoparticles carrying clinically relevant antigens and adjuvants were coated with different DC-SIGN ligands including biotin-ICAM3-Fc fusion proteins. Comparative studies have shown that despite stronger affinity of some antibodies for DC-SIGN, ICAM3-Fc induced more efficient cross-presentation in certain contexts, highlighting the importance of ligand selection in designing DC-SIGN targeted vaccines for clinical applications .

How can researchers assess the functional impact of ICAM3 blocking in neutrophil phagocytosis assays?

To assess the functional impact of ICAM3 blocking on neutrophil phagocytosis:

• Apoptotic Cell Preparation: Induce apoptosis in target cells (typically leukocytes) using standardized methods such as UV irradiation or serum starvation.

• Blocking Protocol:

  • Pre-incubate phagocytes (macrophages) with biotinylated ICAM3 antibody at 5-10 μg/ml

  • Include appropriate controls: isotype control, blocking with unconjugated antibody, and untreated cells

• Phagocytosis Assay Setup:

  • Label apoptotic cells with fluorescent dyes (e.g., CFSE)

  • Co-culture with treated/untreated macrophages at appropriate ratios

  • Allow phagocytosis to occur (typically 1-2 hours)

• Analysis Methods:

  • Flow cytometry: Detect macrophages that have engulfed fluorescent apoptotic cells

  • Microscopy: Visualize and quantify phagocytosis events

  • Biochemical assays: Measure markers of phagosome maturation

Research has established that ICAM3, in association with integrin alpha-L/beta-2, contributes significantly to apoptotic neutrophil phagocytosis by macrophages . Blocking ICAM3 with specific antibodies can disrupt this process, providing insights into the molecular mechanisms of efferocytosis and its immunological consequences.

What strategies can resolve epitope masking issues when using biotin-ICAM3 antibodies in complex tissue samples?

When encountering epitope masking issues in complex tissue samples:

• Antigen Retrieval Optimization:

  • Heat-induced epitope retrieval: Test different buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Enzymatic retrieval: Try proteinase K, trypsin, or pepsin at varying concentrations and incubation times

  • Combination approaches: Sequential application of heat and enzymatic methods

• Fixation Modifications:

  • Reduce fixation time or fixative concentration

  • Compare cross-linking fixatives (paraformaldehyde) with precipitating fixatives (ethanol, methanol)

  • Consider light fixation followed by post-fixation after antibody binding

• Detection System Enhancement:

  • Use tyramide signal amplification with biotinylated antibodies

  • Apply multi-layered detection: biotin-streptavidin-biotin amplification

  • Consider proximity ligation assays for detecting protein-protein interactions involving ICAM3

• Co-staining Sequence Optimization:

  • Test different antibody incubation sequences

  • Apply ICAM3 antibody first in sequential staining protocols

  • Consider section pretreatment with blocking antibodies against potentially interfering proteins

Since the MEM-171 clone recognizes an extracellular epitope in the D2 domain of ICAM3, careful preservation of the protein's tertiary structure is particularly important . When studying ICAM3 in tissue sections containing Langerhans cells or infiltrating leukocytes, these optimization strategies can significantly improve specific detection while minimizing background.

How can researchers address high background when using biotin-conjugated ICAM3 antibodies?

High background is a common challenge with biotin-conjugated antibodies. Here are methodological approaches to address this issue:

• Endogenous Biotin Blocking:

  • Pre-block with avidin followed by biotin (Avidin/Biotin Blocking Kit)

  • Apply streptavidin followed by biocytin blocking

  • For tissues with extremely high endogenous biotin (liver, kidney), consider alternative conjugates

• Buffer Optimization:

  • Increase protein concentration in blocking buffer (3-5% BSA or normal serum)

  • Add 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 to reduce non-specific binding

  • Include 5-10% serum from the species of the secondary reagent

• Antibody Titration:

  • Perform careful titration of the biotinylated ICAM3 antibody (starting range: 1-10 μg/ml)

  • Test different concentrations of streptavidin detection reagent

  • Optimize incubation times and temperatures

• Washing Protocol Enhancement:

  • Increase wash duration and volume

  • Add extra wash steps between critical incubations

  • Include 0.05% Tween-20 in wash buffers

These approaches should be systematically tested and documented to establish optimal conditions for different experimental systems and sample types .

What strategies can resolve discrepancies in ICAM3 detection between different experimental techniques?

When facing discrepancies in ICAM3 detection across different techniques:

• Epitope Accessibility Analysis:

  • The MEM-171 clone recognizes a specific extracellular epitope in the D2 domain of ICAM3

  • Different techniques (flow cytometry vs. immunohistochemistry) may affect epitope exposure differently

  • Test multiple ICAM3 antibody clones recognizing distinct epitopes (MEM-171, MEM-04, 2D11D1) to confirm results

• Sample Processing Comparison:

  • Fresh vs. frozen vs. fixed samples may yield different results

  • Systematically compare detection in matched samples processed through different workflows

  • Document and standardize sample handling procedures to minimize variability

• Cross-validation Approach:

  • Employ orthogonal detection methods (protein level: flow cytometry, Western blot; RNA level: qPCR, RNA-seq)

  • Use genetic manipulation (knockdown, overexpression) to confirm specificity

  • Compare results from different conjugates of the same antibody clone

• Validation Table Example:

By systematically analyzing detection discrepancies, researchers can develop a more comprehensive understanding of ICAM3 biology and expression patterns across different contexts .

How can competitive binding assays be designed to study ICAM3 interactions using biotinylated antibodies?

Competitive binding assays can provide valuable insights into ICAM3 interactions:

• Flow Cytometry-Based Competition:

  • Label ICAM3-expressing cells with constant concentration of biotinylated ICAM3 antibody

  • Add competing ligands (e.g., recombinant DC-SIGN) at varying concentrations

  • Analyze shifts in binding curves to determine competition efficiency

  • This approach has been used to study how ICAM3 functions as a ligand for leukocyte adhesion protein LFA-1 and DC-SIGN

• Biolayer Interferometry Applications:

  • Immobilize biotinylated ICAM3 antibody on streptavidin biosensors

  • Expose to ICAM3 protein to create antibody-antigen complex

  • Challenge with potential binding partners

  • Monitor association/dissociation kinetics in real-time

• Data Analysis Considerations:

  • Calculate IC50 values for different competitors

  • Determine if competition is complete (single binding site) or partial (multiple binding sites)

  • Consider allosteric effects where competitor may alter antibody binding without directly competing

These methods have been instrumental in understanding how ICAM3 participates in the initial contact between dendritic cells and T cells that support primary immune responses, and how it contributes to apoptotic neutrophil phagocytosis by macrophages .

How can biotinylated ICAM3 antibodies contribute to the development of targeted nanoparticle vaccines?

Biotinylated ICAM3 antibodies offer significant potential for targeted nanoparticle vaccine development:

• DC-SIGN Targeting Strategy:

  • ICAM3 is a natural ligand for DC-SIGN on dendritic cells, making ICAM3-targeted approaches biologically relevant

  • Biotinylated ICAM3 antibodies can be conjugated to streptavidin-coated nanoparticles carrying antigens and adjuvants

  • This approach enables specific targeting to dendritic cells to enhance antigen presentation and immune activation

• Comparative Targeting Efficiency:

  • Research has compared nanoparticle vaccines carrying clinically relevant antigens and adjuvants that were coated with different DC-SIGN ligands

  • Despite stronger affinity of some antibodies for DC-SIGN, ICAM3-Fc fusion proteins induced more efficient cross-presentation in certain contexts

  • This highlights the importance of both binding affinity and functional outcomes in designing targeted vaccines

• Advantages of Antibody-Based Targeting:

  • Precise epitope targeting compared to whole protein approaches

  • Greater stability in biological fluids

  • Potential for engineering antibody properties (affinity, Fc interactions)

  • Reduced immunogenicity compared to xenogeneic proteins

These approaches are particularly relevant for developing vaccines targeting dendritic cells to enhance primary immune responses against challenging pathogens or tumors .

What considerations are important when using biotinylated ICAM3 antibodies in multiplex imaging technologies?

When incorporating biotinylated ICAM3 antibodies into multiplex imaging:

Since ICAM3 is expressed on specific immune cell populations (leukocytes, Langerhans cells) but not others (platelets, erythrocytes), it serves as a valuable marker in multiplex imaging strategies for immunophenotyping and spatial analysis of immune cell interactions .

How can researchers validate antibody specificity when studying rare ICAM3 variants or post-translational modifications?

Validating antibody specificity for rare ICAM3 variants requires rigorous methodological approaches:

• Genetic Validation Strategies:

  • Use CRISPR/Cas9 to generate ICAM3 knockout cells as negative controls

  • Create expression constructs for specific ICAM3 variants or mutants

  • Compare detection patterns between wild-type and variant ICAM3

• Biochemical Validation Approach:

  • Immunoprecipitate ICAM3 from relevant samples

  • Perform mass spectrometry to identify the exact protein species being detected

  • Use Western blotting with multiple antibody clones recognizing different epitopes

  • Compare results between biotinylated and non-biotinylated versions of the same antibody clone

• Post-translational Modification Analysis:

  • For glycosylation variants: Compare antibody binding before and after treatment with specific glycosidases

  • For phosphorylation studies: Use phosphatase treatments as controls

  • Combine with phospho-specific antibodies to correlate modifications with epitope accessibility

The MEM-171 clone's specificity for an extracellular epitope in the D2 domain of ICAM3 makes it particularly useful for discriminating between ICAM family members, as this region contains sequence variations that distinguish ICAM3 from other family members .