PECAM1 Recombinant Monoclonal Antibody

What is PECAM1/CD31 and why is it important in research applications?

PECAM1 (Platelet Endothelial Cell Adhesion Molecule-1), also known as CD31, is a transmembrane glycoprotein belonging to the immunoglobulin supergene family of adhesion molecules. This 130-kDa protein is critically important in research due to its widespread expression on hematopoietic stem cells and vascular endothelial cells, making it an invaluable marker for these cell types in experimental studies. PECAM1 plays fundamental roles in multiple cellular functions, including leukocyte transendothelial migration, serving as an anti-apoptotic protein that protects against Bax-mediated cell death, and functioning as part of a shear-dependent mechanosensory complex in vascular endothelium. Its ability to form homophilic interactions allows it to concentrate at endothelial cell intercellular junctions through "diffusion trapping," where it contributes significantly to barrier function and control of vascular permeability .

How do researchers distinguish between different domains of PECAM1 using monoclonal antibodies?

Researchers can differentiate between PECAM1 domains using domain-specific monoclonal antibodies that target distinct regions of the protein. Several well-characterized antibodies have been developed for this purpose, including: (1) antibodies specific for the N-terminal immunoglobulin domain 1 (IgD1), such as PECAM-1.3; (2) antibodies recognizing the membrane-proximal immunoglobulin domain 6 (IgD6), such as PECAM-1.2; and (3) antibodies that bind to the cytoplasmic domain, like mAb 235.1, which recognizes the C-terminal 15 amino acids. These domain-specific antibodies enable researchers to study the structure-function relationships of PECAM1 and have been instrumental in understanding how different domains contribute to the protein's varied biological functions. Experimental validation of domain specificity is typically performed using ELISA analysis with purified PECAM1 as the target antigen or by evaluating binding to PECAM1-expressing cells using flow cytometry and immunofluorescence microscopy .

What are the standard methods for validating PECAM1 antibody specificity in experimental protocols?

Validating PECAM1 antibody specificity is crucial for generating reliable research data. Standard methods include:

• Cell-bound ELISA: Cells expressing PECAM1 are fixed, blocked, and incubated with the antibody of interest, followed by detection with a secondary antibody conjugated to an enzyme for colorimetric detection. This method allows quantification of binding affinity and specificity.

• Competition ELISA: This involves mixing serial dilutions of a known anti-PECAM1 antibody with the test antibody and measuring competitive binding to PECAM1-expressing cells, which confirms epitope specificity.

• Flow cytometry: Antibodies are tested for their ability to bind PECAM1-expressing cells versus control cells, providing data on both specificity and relative binding strength.

• Immunofluorescence microscopy: This visualizes the pattern of antibody binding to confirm proper localization to cell junctions where PECAM1 is known to concentrate.

• Western blotting: This verifies that the antibody recognizes a protein of the expected molecular weight.

Additional validation should include testing across multiple PECAM1-expressing cell types and comparing binding patterns with established reference antibodies .

How can PECAM1 antibodies be utilized to study endothelial barrier function in inflammatory conditions?

PECAM1 antibodies provide sophisticated tools for investigating endothelial barrier function during inflammatory challenges. Researchers can employ these antibodies in Electric Cell-substrate Impedance Sensing (ECIS) assays to measure real-time changes in barrier integrity. The experimental approach involves:

• Growing endothelial cells to confluence on gold electrodes coated with fibrinogen

• Establishing baseline barrier measurements via electrical resistance

• Introducing inflammatory stimuli (e.g., thrombin) to disrupt the endothelial barrier

• Applying anti-PECAM1 antibodies (typically as Fab fragments at 40 μg/ml) during barrier recovery

• Continuously monitoring electrical resistance to quantify barrier restoration

This system allows researchers to determine how antibodies binding to specific PECAM1 domains affect barrier recovery following inflammatory challenge. Studies have demonstrated that antibodies targeting different domains of PECAM1 can either enhance or impair barrier restoration, depending on which domain they bind. For example, antibodies that modulate the adhesive properties of PECAM1 can significantly increase the rate of barrier recovery after inflammatory disruption. These assays provide valuable quantitative data expressed as average basal electrical resistances (in Ω/cm²), allowing statistical comparison between different antibody treatments and control conditions .

What methodological approaches can be used to investigate PECAM1-mediated cell migration using recombinant monoclonal antibodies?

Investigating PECAM1-mediated cell migration using recombinant monoclonal antibodies requires specialized methodological approaches. One sophisticated technique involves the Electric Cell-substrate Impedance Sensing (ECIS) wound healing assay:

• Endothelial cells are grown to confluence on 8W1E electrode arrays

• A defined elevated field pulse (1400 mA, 40000 Hz, 14 s) creates a precise injury area

• Cell migration into the electrically wounded area is measured in real-time

• Anti-PECAM1 antibodies (typically 40 μg/ml of Fab fragments) are introduced to assess their effects on migration rates

Alternative approaches include:

• Transwell migration assays with PECAM1 antibody-coated membranes

• Time-lapse microscopy tracking of individual cell movements following antibody treatment

• 3D migration assays in collagen matrices with PECAM1 antibody supplementation

Data analysis should include quantification of migration rates, directional persistence, and cellular morphology changes. Statistical analysis typically employs one-way ANOVA followed by Bonferroni's multiple-comparisons test, with significance threshold set at p < 0.05. This methodology allows researchers to precisely determine how antibodies targeting different PECAM1 epitopes influence endothelial cell migration, which has implications for understanding angiogenesis and vascular repair mechanisms .

How do researchers design fusion constructs of anti-PECAM1 antibodies for targeted drug delivery applications?

Designing fusion constructs of anti-PECAM1 antibodies for targeted drug delivery requires a sophisticated approach to molecular engineering. Based on established research protocols, the process typically involves:

• Selection of antibody fragment format: Single-chain variable fragments (scFv) derived from anti-PECAM1 monoclonal antibodies are commonly used due to their smaller size and retention of binding specificity. These can be generated by:

  • PCR amplification of variable heavy and light chain regions from hybridoma cDNA

  • Assembly using a flexible linker sequence such as (Gly₄Ser)₃ to maintain proper protein folding

• Therapeutic cargo selection: Various therapeutic molecules can be fused to anti-PECAM1 scFv, including:

  • Enzymes like low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA)

  • Cytokines or growth factors

  • Drug-loaded nanocarriers

• Linker design: The linker between the scFv and therapeutic cargo is critical for maintaining function of both components:

  • Flexible linkers like (Ser₄Gly)₂Ala₃ are commonly used

  • Cleavable linkers may be incorporated for controlled release

• Expression vector construction: This typically involves:

  • Introducing appropriate restriction sites (e.g., SpeI, NotI, XhoI) for cloning

  • Assembling the construct using overlap extension PCR

  • Inserting the fusion construct into expression vectors like pMT/Bip/V5 for eukaryotic expression

The final construct must be validated for both targeting specificity and therapeutic functionality. Typical yields from optimized expression systems like S2 Drosophila cells are approximately 5 mg/L of purified fusion protein .

What factors should researchers consider when selecting fluorophore conjugates for PECAM1 antibodies in imaging applications?

When selecting fluorophore conjugates for PECAM1 antibodies in imaging applications, researchers should consider several critical factors that impact experimental outcomes:

• Fluorophore spectral properties:

  • Excitation/emission profiles should match available microscopy filter sets and laser lines

  • Consider potential spectral overlap with other fluorophores when designing multi-color experiments

• Brightness and photostability:

  • CF® dyes offer exceptional brightness and photostability compared to conventional fluorophores

  • For long-term imaging experiments, photostability is particularly important to prevent signal loss

• Target abundance considerations:

  • Blue fluorescent dyes (e.g., CF®405S, CF®405M) are not recommended for detecting low-abundance PECAM1 expression due to:
    a) Lower fluorescence intensity
    b) Higher non-specific background compared to other dye colors

  • For low expression systems, brighter dyes in the green to far-red spectrum are preferable

• Detection channel compatibility:

  Fluorophore	Ex/Em (nm)	Laser line	Compatible channel
  CF®405S	404/431	405 nm	DAPI (microscopy), AF405
  CF®488A	490/515	488 nm	GFP, FITC
  CF®568	562/583	532, 561 nm	RFP, TRITC

• Background considerations:

  • Autofluorescence in the blue-green spectrum can interfere with detection

  • Far-red dyes may provide better signal-to-noise ratios in tissues with high autofluorescence

These considerations enable researchers to optimize detection sensitivity and specificity for PECAM1 visualization in various experimental contexts .

What are the optimal sample preparation methods for electron microscopy studies of PECAM1 structure and distribution?

Optimal sample preparation for electron microscopy studies of PECAM1 structure and distribution requires meticulous attention to detail to preserve native protein conformation. Based on established protocols from specialized facilities like the Brookhaven National Laboratory, the recommended methodology includes:

• Specimen dilution and preparation:

  • Dilute purified PECAM1 extracellular domain to 10 μg/ml in physiological buffer (0.1 M NaCl, 20 mM HEPES, pH 7.0)

  • Apply 3 μl aliquots to carbon-film coated microscope grids

  • Allow 1-minute attachment time for optimal protein distribution

• Buffer exchange and cryogenic preparation:

  • Exchange grid surface fluid 8-10 times with 150 mM ammonium acetate solution to remove salts

  • Flash-freeze specimens in liquid nitrogen to preserve native structure

  • Perform freeze-drying under vacuum conditions to prevent ice crystal formation

• Imaging parameters for Scanning Transmission Electron Microscopy (STEM):

  • Use uncontrasted specimens to visualize the natural protein density

  • Employ a 40-kV probe focused at 0.25 nm for high-resolution imaging

  • Verify monomolecular or bimolecular forms through mass analysis of individual objects

• Data analysis considerations:

  • Perform quantitative mass measurements to determine molecular weight

  • Evaluate structural features such as domain organization and conformation

  • Compare experimental images with predicted models based on amino acid sequence

This methodology enables researchers to visualize PECAM1 structural features at molecular resolution, providing insights into how antibody binding might affect protein conformation and function .

How should researchers optimize antibody concentration and incubation conditions for PECAM1 detection in different experimental systems?

Optimizing antibody concentration and incubation conditions for PECAM1 detection requires systematic approach across different experimental systems. Based on the research literature, the following methodological guidelines should be implemented:

• For cell-bound ELISA detection systems:

  • Perform initial titration experiments using serial dilutions (typically 0.1-50 μg/ml) of biotinylated fusion proteins

  • Determine optimal concentration based on signal-to-background ratio

  • Cell fixation with ice-cold methanol provides optimal epitope exposure

  • Block with 5% BSA/PBS containing 1 μg/ml scuPA to prevent non-specific binding

  • Detection with peroxidase-conjugated streptavidin and OPD substrate provides sensitive colorimetric readout

  • Absorbance measurement at 490 nm provides quantitative assessment of binding

• For competition binding assays:

  • Use 20 μg/ml of the fusion protein as standard concentration

  • Mix with serial dilutions of competing antibody before cell application

  • Calculate IC50 values to determine relative binding affinities

• For functional barrier integrity experiments:

  • Optimal antibody concentration is typically 40 μg/ml of Fab fragments

  • Apply antibodies at the nadir of resistance following thrombin- or electrical-induced injury

  • Monitor real-time changes in barrier function parameters

• Statistical validation of optimization:

  • Analyze data using one-way ANOVA followed by Bonferroni's multiple-comparisons test

  • Establish significant differences with p < 0.05 threshold

  • Express results as mean ± standard deviation to account for experimental variability

These optimization guidelines ensure consistent, reproducible results across different experimental platforms while maximizing sensitivity and specificity of PECAM1 detection .

How can PECAM1 antibodies be used to assess tumor angiogenesis and potentially predict tumor recurrence?

PECAM1 antibodies provide critical tools for quantitative assessment of tumor angiogenesis with potential prognostic value. The methodological approach involves:

• Tissue preparation and antibody selection:

  • Formalin-fixed, paraffin-embedded tumor sections are typically used

  • High-specificity recombinant monoclonal anti-CD31/PECAM1 antibodies are preferred over polyclonal antibodies for reproducibility

  • Antibody clones with validated specificity for endothelial cells (e.g., C31/1395R) provide optimal results

• Quantification methodology:

  • Microvessel density (MVD) determination: Count PECAM1-positive vessels in high-power fields (typically 200-400×) in areas of highest vascularization ("hot spots")

  • Measure vessel diameter, area, and perimeter using computerized image analysis

  • Evaluate vessel maturation by co-staining with pericyte markers (e.g., α-SMA)

• Expression level assessment:

  • Quantify PECAM1 staining intensity using digital image analysis (0-3+ scale)

  • Measure percentage of PECAM1-positive area within the tumor section

  • Calculate H-score (intensity × percentage) for standardized comparison

• Correlation with clinical outcomes:

  • High levels of PECAM1 expression often correlate with rapidly growing tumors

  • Elevated microvessel density may serve as a predictor of tumor recurrence

  • Statistical analysis should adjust for tumor type, stage, and other prognostic factors

When implementing this methodology, researchers should be aware that PECAM1 staining of non-vascular tumors (excluding hematopoietic neoplasms) is rare, making it a reliable marker for distinguishing tumor vasculature from malignant cells themselves. This approach enables quantitative assessment of angiogenesis as both a biological feature and potential prognostic indicator .

What methodological approaches can be used to develop PECAM1-targeted therapeutics for vascular permeability disorders?

Developing PECAM1-targeted therapeutics for vascular permeability disorders requires sophisticated methodological approaches that leverage the protein's unique properties. Based on current research, the following methodological framework is recommended:

• Regulatable PECAM1-targeting strategy development:

  • Utilize the finding that PECAM1's adhesive properties can be modulated by antibodies binding to specific domains

  • Design antibodies or antibody fragments that enhance PECAM1 homophilic interactions to strengthen endothelial barriers

  • Target membrane-proximal IgD6 domain, which has been shown to regulate adhesive interactions

• Nanodisc technology implementation:

  • Incorporate full-length, monomeric PECAM1 into phosphatidylcholine-containing nanodiscs

  • Verify that PECAM1-containing nanodiscs retain IgD1-dependent homophilic binding capabilities

  • Test the ability of domain-specific antibodies to modulate these interactions

• Functional validation methodology:

  • Use Electric Cell-substrate Impedance Sensing (ECIS) to measure endothelial barrier function in real-time

  • Apply inflammatory challenges (e.g., thrombin) to disrupt barriers

  • Evaluate the ability of PECAM1-targeted therapeutics to enhance barrier restoration

  • Quantify results using the barrier function parameter (Rb), expressed as average basal electrical resistances

• Translational development approach:

  • Design fusion proteins combining PECAM1-targeting domains with therapeutic effectors

  • For instance, create constructs that fuse anti-PECAM1 scFv with stabilizers of endothelial junctions

  • Evaluate in vitro efficacy before advancing to animal models of vascular leak

This methodological framework provides a scientific basis for developing novel therapeutics that can control endothelial cell barrier function in various vascular permeability disorders, potentially addressing conditions like acute respiratory distress syndrome, septic shock, and ischemia-reperfusion injury .

How can researchers address non-specific binding issues when using PECAM1 antibodies in immunohistochemistry and flow cytometry?

Non-specific binding is a common challenge when working with PECAM1 antibodies in various detection methods. To address this issue systematically, researchers should implement the following methodological approaches:

• For immunohistochemistry applications:

  • Optimize blocking conditions using 5% BSA in PBS with added 1 μg/mL scuPA (single-chain urokinase plasminogen activator) to reduce background

  • Perform antigen retrieval optimization: Compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0) to determine which best exposes PECAM1 epitopes while minimizing non-specific binding

  • Titrate primary antibody concentration using serial dilutions (typically 1-10 μg/mL) to identify optimal signal-to-noise ratio

  • Include isotype control antibodies at matching concentrations to distinguish specific from non-specific binding

  • Consider using Fab fragments instead of whole IgG to reduce Fc receptor-mediated binding

• For flow cytometry applications:

  • Include viability dye (e.g., 7-AAD or LIVE/DEAD™ fixable dyes) to exclude dead cells, which often bind antibodies non-specifically

  • Block Fc receptors before antibody staining using commercial Fc block or 10% serum from the same species as the secondary antibody

  • Evaluate fluorophore-specific autofluorescence by analyzing unstained samples in all detection channels

  • When using blue fluorescent dyes (e.g., CF®405S), be particularly vigilant as these conjugates show higher non-specific background than other dye colors

• Validation controls to implement:

  • Perform competition assays with unlabeled antibody to confirm specificity

  • Include PECAM1-negative cell lines as negative controls

  • Use multiple anti-PECAM1 antibodies targeting different epitopes to confirm staining patterns

By systematically implementing these approaches, researchers can significantly reduce non-specific binding issues and generate more reliable and reproducible results with PECAM1 antibodies .

What are the critical quality control parameters for evaluating new batches of PECAM1 recombinant monoclonal antibodies?

Evaluating new batches of PECAM1 recombinant monoclonal antibodies requires rigorous quality control to ensure experimental reproducibility. The following critical parameters should be assessed:

• Binding specificity verification:

  • Perform ELISA against purified human platelet PECAM1 as target antigen

  • Conduct flow cytometry analysis using known PECAM1-positive cell lines (e.g., endothelial cells) and negative control cell lines

  • Implement competition binding assays with established reference antibodies

  • Verify epitope specificity through domain-specific binding tests against:

    • N-terminal IgD1 domain

    • Membrane-proximal IgD6 domain

    • Cytoplasmic domain (if applicable)

• Functional activity assessment:

  • Evaluate the antibody's ability to modulate PECAM1-dependent functions:

    • Barrier function in endothelial monolayers

    • Cell migration in wound healing assays

    • Leukocyte transendothelial migration (if relevant to application)

  • Compare functional effects to reference antibody batches

• Physical and biochemical characterization:

  • Confirm protein concentration using standardized methods (BCA or Bradford assay)

  • Verify antibody purity by SDS-PAGE (>95% purity expected)

  • Assess aggregation status using dynamic light scattering or size exclusion chromatography

  • For fluorophore-conjugated antibodies, determine dye-to-protein ratio:

    Antibody # prefix	Conjugation	Optimal dye-to-protein ratio
    BNC04	CF®405S	2-4
    BNC88	CF®488A	4-6
    BNC68	CF®568	2-4

• Stability testing:

  • Evaluate binding activity after multiple freeze-thaw cycles

  • Perform accelerated stability studies at elevated temperatures

  • Assess long-term storage stability at recommended conditions

Implementing these quality control measures ensures that each new batch of PECAM1 recombinant monoclonal antibody meets the required specifications for research applications, minimizing batch-to-batch variability .