ICAM2 Antibody

What is ICAM2 and what biological functions does it serve?

ICAM2 (Intercellular Adhesion Molecule 2), also known as CD102, is a glycosylated cell surface protein that belongs to the immunoglobulin superfamily. It is broadly expressed on most leukocytes and is strongly expressed by endothelial cells, with a molecular weight of approximately.30.7 kilodaltons . ICAM2 functions as a ligand for the leukocyte adhesion protein LFA-1 (Lymphocyte Function-associated Antigen-1, composed of integrin alpha-L/beta-2) . The protein plays critical roles in several immunological processes, including lymphocyte recirculation by potentially blocking LFA-1-dependent cell adhesion. Additionally, ICAM2 mediates adhesive interactions important for antigen-specific immune responses, natural killer (NK) cell-mediated clearance, and other cellular interactions crucial for immune response and surveillance mechanisms . Understanding these functions is essential for researchers designing experiments targeting immune cell trafficking and activation pathways.

What are the primary applications for ICAM2 antibodies in research?

ICAM2 antibodies are utilized across multiple experimental applications, with the most common being Flow Cytometry (FCM), Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), and Enzyme-Linked Immunosorbent Assay (ELISA) . Flow cytometry represents a particularly validated application, with specific dilution recommendations available (e.g., 1:100 dilution, using 10μl to label 10^6 cells in 100μl) . Some clones, such as CBRIC2/2, are specifically reported to inhibit interactions between ICAM2 and LFA-1, making them valuable for functional blocking studies in addition to detection applications . Researchers should select antibodies with validation data for their specific application to ensure reliable results in their experimental systems.

What species reactivity is available for ICAM2 antibodies?

The market offers ICAM2 antibodies with reactivity against multiple species. Human-reactive antibodies are most common, with numerous validated clones available including B-T1 and CBRIC2/2 . Mouse-reactive antibodies are also available, with some suppliers offering specific anti-mouse CD102 antibodies . Additionally, some antibodies demonstrate cross-reactivity with rat models . Based on gene sequence homology, researchers working with canine, porcine, and non-human primate models may find suitable antibodies, though validation in these species may be necessary . When selecting antibodies for non-human models, researchers should carefully review validation data or consider performing preliminary validation studies to confirm reactivity in their experimental system.

What are the available formats and conjugations of ICAM2 antibodies?

ICAM2 antibodies are available in multiple formats to suit different experimental needs. The primary formats include:

• Unconjugated antibodies - These provide flexibility for custom detection strategies and are the most common format

• Conjugated antibodies - Available with various tags including:

  • Fluorescent labels: Cy3, DyLight488

  • Enzymatic labels: Biotin

  • Other specialized tags: Available upon request from some suppliers

The choice between formats depends on the experimental design, with unconjugated antibodies offering versatility for secondary detection systems, while directly conjugated antibodies reduce protocol steps and may improve signal-to-noise ratios in certain applications like flow cytometry and microscopy.

What are the optimal storage conditions for maintaining ICAM2 antibody activity?

Proper storage is critical for maintaining antibody functionality. ICAM2 antibodies are typically shipped at ambient temperature, but long-term storage requires specific conditions . For optimal preservation, antibodies should be aliquoted upon receipt and stored at -20°C for long-term stability . For short-term use (up to 4 weeks), storage at 4°C is acceptable . Multiple freeze-thaw cycles should be strictly avoided as they can degrade antibody performance, which is why creating single-use aliquots upon receipt is highly recommended . Additionally, storage in frost-free freezers is not recommended due to the temperature fluctuations that occur during auto-defrost cycles . For antibodies in liquid formulation, researchers should check if the buffer contains preservatives like sodium azide (commonly at 0.09% concentration), which helps prevent microbial contamination during storage .

How should researchers validate the specificity of ICAM2 antibodies?

Validating antibody specificity is crucial for generating reliable data. For ICAM2 antibodies, several validation approaches are recommended:

• Positive and negative controls: Use cell lines known to express or lack ICAM2 expression. Endothelial cells serve as excellent positive controls due to their strong ICAM2 expression .

• Blocking experiments: Some antibody clones like CBRIC2/2 can block ICAM2-LFA-1 interactions, which can be used as a functional validation approach .

• Knockout/knockdown validation: Compare antibody staining between wildtype cells and those with ICAM2 genetically depleted.

• Recombinant protein controls: Test antibody binding against purified ICAM2 protein versus irrelevant proteins.

• Cross-application validation: Confirm consistent protein detection across multiple applications (e.g., flow cytometry and Western blot) to strengthen confidence in specificity.

Thorough validation ensures experimental results accurately reflect ICAM2 biology rather than non-specific binding artifacts.

What are the optimal experimental conditions for using ICAM2 antibodies in flow cytometry?

Flow cytometry represents one of the most common applications for ICAM2 antibodies, particularly for analyzing expression on leukocytes and endothelial cells. For optimal results, researchers should consider the following protocol parameters:

• Antibody dilution: A typical working dilution of 1:100 is recommended for flow cytometry applications .

• Cell concentration: Use approximately 10μl of diluted antibody solution to label 10^6 cells in 100μl of buffer .

• Buffer composition: Phosphate-buffered saline containing 1-2% protein (BSA or FBS) and 0.05% sodium azide is typically suitable for flow cytometry staining.

• Incubation conditions: A 30-60 minute incubation at 4°C in the dark is standard for surface staining of ICAM2.

• Washing steps: At least two washes with excess buffer after antibody incubation will reduce background signal.

• Secondary detection: For unconjugated primary antibodies, appropriate species and isotype-matched secondary antibodies should be used, such as goat anti-mouse APC conjugates for mouse monoclonal primaries .

• Controls: Include appropriate isotype controls matching the primary antibody's host species and isotype (e.g., mouse IgG2a for the CBRIC2/2 clone) .

Optimizing these parameters will ensure high signal-to-noise ratios and accurate detection of ICAM2-expressing populations.

How can ICAM2 antibodies be used to investigate lymphocyte trafficking mechanisms?

ICAM2 plays a critical role in lymphocyte recirculation through its interaction with LFA-1 on leukocytes . Researchers can leverage ICAM2 antibodies to investigate these trafficking mechanisms through several experimental approaches:

• Adhesion blocking studies: Antibodies like clone CBRIC2/2 are reported to inhibit interactions between ICAM2 and LFA-1, allowing researchers to assess the functional contribution of ICAM2 to cell adhesion in vitro and potentially in vivo .

• Flow-based adhesion assays: Researchers can coat flow chambers with recombinant ICAM2 and use blocking antibodies to quantify the specific contribution of ICAM2 to lymphocyte adhesion under physiological shear stress conditions.

• Intravital microscopy: When combined with fluorescent labeling techniques, ICAM2 antibodies can help visualize the dynamics of lymphocyte-endothelial interactions in living tissues.

• Transendothelial migration assays: ICAM2 antibodies can be used to determine the role of this adhesion molecule in the multi-step process of leukocyte extravasation from blood vessels into tissues.

• Adoptive transfer experiments: Pretreatment of cells with blocking ICAM2 antibodies before transfer into recipient animals can help elucidate the role of ICAM2 in homing to specific tissues.

These approaches can provide mechanistic insights into how ICAM2 contributes to normal lymphocyte trafficking and how dysregulation may contribute to inflammatory and immune-mediated diseases.

What are the considerations for using ICAM2 antibodies in immunohistochemistry?

Immunohistochemistry (IHC) applications for ICAM2 antibodies require specific optimization, particularly when studying endothelial expression patterns. Researchers should consider:

• Fixation sensitivity: Many ICAM2 epitopes are sensitive to fixation methods. Fresh-frozen tissue sections (IHC-Fr) are often preferred over formalin-fixed paraffin-embedded (FFPE) samples for ICAM2 detection .

• Antigen retrieval: If using FFPE tissues, optimization of antigen retrieval methods is critical, with citrate buffer (pH 6.0) often providing better results than EDTA-based buffers.

• Blocking conditions: Thorough blocking of endogenous peroxidase activity and non-specific binding sites is essential, particularly when studying ICAM2 in highly vascularized tissues.

• Detection systems: Amplification systems like tyramide signal amplification may improve detection of low-abundance ICAM2 expression in some tissues.

• Counterstaining: Combining ICAM2 staining with endothelial markers (CD31, vWF) can help distinguish ICAM2 expression on endothelial cells versus infiltrating leukocytes.

• Quantification: Digital image analysis using specialized software can provide objective quantification of ICAM2 expression across different experimental conditions.

Optimizing these parameters is crucial for accurate visualization and quantification of ICAM2 distribution in tissues for studies of vascular biology and inflammation.

How can researchers troubleshoot common issues with ICAM2 antibody experiments?

When working with ICAM2 antibodies, researchers may encounter several common challenges. Here are troubleshooting strategies for addressing these issues:

• Weak or no signal:

  • Verify ICAM2 expression levels in your sample type

  • Increase antibody concentration or incubation time

  • Try alternative antibody clones with different epitope recognition

  • Ensure proper storage conditions have been maintained

  • For flow cytometry, check that cells are viable and surface molecules are preserved

• High background:

  • Implement more stringent blocking steps

  • Reduce primary and/or secondary antibody concentrations

  • Include additional washing steps

  • Use appropriate isotype controls to identify non-specific binding

  • For IHC, optimize fixation and antigen retrieval conditions

• Inconsistent results between experiments:

  • Standardize protocols with detailed SOPs

  • Use consistent cell densities and passage numbers

  • Prepare fresh antibody dilutions for each experiment

  • Include positive control samples in each experiment

  • Consider using automated systems to reduce operator variability

• Cross-reactivity issues:

  • Verify antibody specificity using knockout/knockdown controls

  • Test multiple antibody clones targeting different epitopes

  • Adjust blocking conditions to reduce non-specific binding

  • Use pre-absorption controls with recombinant proteins

Systematic troubleshooting using these approaches can help resolve technical issues and generate reliable, reproducible data in ICAM2 research.

How are ICAM2 antibodies being used in cancer immunotherapy research?

ICAM2 has emerging roles in cancer biology and immunotherapy research. Researchers are exploring several innovative applications:

• Tumor vasculature targeting: Since ICAM2 is strongly expressed on endothelial cells, antibodies can be used to study tumor angiogenesis and potentially develop vascular-targeting therapies.

• Immune checkpoint modulation: The interaction between ICAM2 and LFA-1 represents a potential immunoregulatory pathway that researchers are investigating for cancer immunotherapy, with blocking antibodies serving as valuable tools.

• Chimeric antigen receptor (CAR) development: ICAM2-targeting single-chain variable fragments derived from antibodies like CBRIC2/2 can be incorporated into CAR constructs for potential targeting of ICAM2-expressing malignancies.

• Antibody-drug conjugates (ADCs): ICAM2 antibodies can be conjugated to cytotoxic payloads for targeted delivery to ICAM2-expressing cells in the tumor microenvironment.

• Imaging agent development: Fluorescently-labeled or radiolabeled ICAM2 antibodies are being explored for cancer imaging applications, particularly for tumors with strong vascular components.

These emerging applications highlight the versatility of ICAM2 antibodies beyond traditional research applications and their potential translation to clinical oncology research.

What are the current limitations of available ICAM2 antibodies and how might they be addressed?

Despite their utility, current ICAM2 antibodies have several limitations that researchers should consider:

• Species cross-reactivity limitations: While human-reactive antibodies are abundant, validated antibodies for some experimental animal models remain limited . To address this, researchers can:

  • Screen antibodies across species using conserved epitopes

  • Develop new antibodies against species-specific ICAM2 variants

  • Use genetic approaches (e.g., humanized mouse models) when antibodies are limiting

• Limited functional characterization: Many commercially available antibodies have been characterized for binding but not for functional effects on ICAM2-mediated processes . Researchers can:

  • Perform functional assays to identify blocking vs. non-blocking antibodies

  • Characterize effects on signaling pathways downstream of ICAM2

  • Develop antibodies targeting specific functional domains

• Epitope information gaps: Detailed epitope mapping is often unavailable, limiting understanding of how antibodies might interfere with specific protein interactions . Solutions include:

  • Conducting epitope mapping studies with existing antibodies

  • Developing epitope-specific antibodies against different ICAM2 domains

  • Using structural biology approaches to characterize antibody-antigen interactions

• Application restrictions: Not all antibodies work across multiple applications, requiring researchers to purchase different clones for different techniques . To address this:

  • Validate antibodies across multiple applications before beginning large studies

  • Develop new multi-purpose antibodies with broader application compatibility

  • Create application-specific derivatives of well-characterized antibody clones

Addressing these limitations will expand the utility of ICAM2 antibodies in both basic research and translational applications.

How is ICAM2 antibody technology likely to evolve in coming years?

The field of ICAM2 antibody research is poised for significant technological advancements. Several trends and developments are anticipated:

• Recombinant antibody development: A shift from hybridoma-derived to recombinant antibody production will likely improve consistency and reduce batch-to-batch variation in ICAM2 antibodies.

• Nanobodies and single-domain antibodies: Development of smaller antibody formats derived from camelid antibodies may offer improved tissue penetration and novel applications for ICAM2 research.

• Multiplex detection systems: Advanced conjugation technologies will enable simultaneous detection of ICAM2 alongside other adhesion molecules and signaling proteins in complex systems.

• AI-assisted antibody design: Computational approaches will accelerate the development of ICAM2 antibodies with improved specificity, affinity, and cross-species reactivity.

• Spatially-resolved antibody applications: Integration with technologies like spatial transcriptomics and imaging mass cytometry will provide unprecedented insights into ICAM2 distribution and function in tissues.

• Humanized antibody derivatives: For translational applications, humanization of effective research antibodies will create new therapeutic candidates targeting ICAM2-mediated pathologies.