CD31 Recombinant Monoclonal Antibody

What is CD31 and why is it significant in vascular research?

CD31 (also known as PECAM-1) is a 130kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules. It functions as an inhibitory coreceptor involved in regulating T cell and B cell signaling through dual immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD31 is expressed ubiquitously within the vascular compartment, primarily at junctions between adjacent endothelial cells, and serves as a specific marker for endothelial differentiation . Its significance in vascular research stems from its multifunctional nature, with diverse roles in integrin-mediated cell adhesion, transendothelial migration of leukocytes, angiogenesis, apoptosis, negative regulation of immunoreceptor signaling, and thrombosis . Additionally, CD31 expression levels can help determine the degree of tumor angiogenesis, with high expression potentially indicating rapidly growing tumors and serving as a predictor of tumor recurrence .

How are CD31 recombinant monoclonal antibodies produced?

CD31 recombinant monoclonal antibodies are produced using in vitro expression systems through a sophisticated process that begins with cloning specific antibody DNA sequences from immunoreactive rabbits or mice. The expression systems are developed specifically for the production of these antibodies, and individual clones undergo rigorous screening to select the best candidates for production . The process typically involves:

• Identification of immunoreactive host animals

• Isolation of B cells producing antibodies against CD31

• Sequencing of antibody-producing genes

• Cloning these sequences into expression vectors

• Transfection into production cell lines

• Screening of resultant antibody clones for specificity and sensitivity

• Large-scale production of selected clones

• Purification and quality control testing

This approach differs significantly from traditional hybridoma techniques and offers numerous advantages for research applications .

What are the key advantages of recombinant monoclonal antibodies for CD31 detection?

Recombinant CD31 monoclonal antibodies offer several distinct advantages over traditional monoclonal antibodies:

• Enhanced specificity and sensitivity: The recombinant production process allows for selection of clones with optimal binding characteristics, resulting in more precise target recognition and reduced background .

• Consistent lot-to-lot performance: Because these antibodies are produced in controlled in vitro systems rather than animals, their production characteristics remain highly stable between manufacturing batches, ensuring more reproducible experimental results .

• Animal origin-free formulations: Many recombinant antibody preparations are completely animal-component free, eliminating concerns about potential contaminants from animal sources and simplifying regulatory compliance for translational research .

• Broader immunoreactivity: The larger immune repertoire of rabbits compared to traditional mouse antibody production systems enables development of antibodies with recognition capabilities across more diverse epitopes and sometimes across multiple species .

• Improved stability: Recombinant antibodies often demonstrate superior shelf-life and stability during storage and experimental use.

Which cellular structures and cell types can be effectively identified using CD31 antibodies?

CD31 antibodies specifically target cell membrane-associated CD31/PECAM-1 and are effective for identifying numerous structures and cell types:

• Endothelial cells: CD31 is highly expressed on endothelial cells lining blood vessels, making these antibodies excellent markers for vascular structures in tissues .

• Endothelial junctions: CD31 is concentrated at intercellular junctions between adjacent endothelial cells .

• Hematopoietic and immune cells: CD31 is expressed on platelets, monocytes, neutrophils, natural killer cells, and certain T-cell populations .

• Vascular tumors: CD31 antibodies can identify both benign and malignant endothelial cells in angiosarcomas with greater consistency than some other vascular markers .

• Tumor blood vessels: These antibodies enable visualization and quantification of tumor vasculature, aiding assessment of angiogenesis in cancer research .

• Lateral border recycling compartment (LBRC): CD31 plays a critical role in this structure, which is essential for leukocyte transendothelial migration .

What methodological considerations are important when using CD31 antibodies for quantitative analysis of tumor angiogenesis?

When employing CD31 antibodies for tumor angiogenesis quantification, researchers should consider several methodological factors to ensure robust and reproducible results:

• Antibody clone selection: Different CD31 antibody clones may have varying sensitivities for detecting tumor vasculature. Clones like JC70 have demonstrated superior capabilities for evaluating tumor angiogenesis compared to some other vascular markers .

• Tissue processing protocol standardization: Fixation time, processing methods, and antigen retrieval techniques must be standardized across all samples to ensure comparable staining intensity and pattern recognition.

• Microvessel density (MVD) quantification approach: Establish clear criteria for what constitutes a countable vessel (e.g., any CD31-positive cell or cell cluster separate from adjacent vessels). The methodology should include:

  • Identification of vascular hotspots at low magnification

  • Vessel counting in multiple high-power fields

  • Averaging of counts across fields and specimens

  • Use of digital image analysis software for objectivity

• Distinction between tumor and normal vasculature: CD31 positivity alone cannot differentiate tumor-associated vessels from pre-existing normal vasculature. Consider combining CD31 with other markers (e.g., αVβ3 integrin) for more specific identification of newly formed tumor vessels.

• Correlation with clinicopathological parameters: The level of CD31 expression can help determine the degree of tumor angiogenesis, and high levels may indicate rapidly growing tumors and potentially predict tumor recurrence .

What are the optimal dilutions and protocols for using CD31 antibodies across different applications?

Optimal protocols for CD31 antibody usage vary by application and specific clone. Based on manufacturer recommendations and research practices:

Western Blotting (WB):

• Recommended dilution range: 1:1000-1:5000

• Expected molecular weight: 130 kDa (note: observed MW may differ from calculated MW of 83 kDa due to post-translational modifications)

• Sample preparation should include membrane fraction enrichment for optimal results

• Verified samples: Jurkat cell lysates

Immunohistochemistry for Paraffin-embedded tissues (IHC-P):

• Recommended dilution range: 1:20-1:100

• Antigen retrieval methods: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

• Verified samples: Human lung cancer tissues

• Incubation time: Typically 30-60 minutes at room temperature or overnight at 4°C

Flow Cytometry:

• Recommended clone for human samples: WM59

• Titration is essential for determining optimal concentration for specific experimental conditions

• Use appropriate isotype controls to establish gating strategies

Immunofluorescence/Immunocytochemistry:

• The WM59 clone has been validated for ICC/IF applications with human samples

• Include appropriate controls to distinguish specific staining from background fluorescence

How can researchers troubleshoot inconsistent CD31 antibody staining in tissue sections?

When encountering inconsistent CD31 staining in tissue sections, consider the following troubleshooting approaches:

• Fixation issues:

  • Over-fixation may mask CD31 epitopes; limit fixation time with formalin to 24-48 hours

  • Under-fixation can lead to tissue degradation and loss of antigenicity

  • Try alternative fixatives if formalin-fixed tissues yield poor results

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (HIER) and enzymatic retrieval methods

  • Vary pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 8.0-9.0)

  • Adjust retrieval time and temperature parameters

• Antibody dilution titration:

  • Perform a systematic dilution series to identify optimal antibody concentration

  • Excessive antibody concentration can increase background staining

  • Too dilute antibody may yield false-negative results

• Block optimization:

  • Use species-appropriate blocking sera to reduce non-specific binding

  • Consider specialized blocking reagents for tissues with high endogenous biotin or peroxidase activity

  • Extend blocking times for problematic tissues

• Detection system selection:

  • Polymer-based detection systems may offer improved sensitivity compared to traditional avidin-biotin systems

  • Tyramide signal amplification can enhance detection of low-abundance CD31 in poorly vascularized tissues

  • Consider fluorescent detection for multiplex applications or when background is problematic

• Tissue-specific considerations:

  • CD31 epitope preservation varies between tissue types; lung and kidney typically show robust staining

  • Necrotic or hypoxic tumor regions may show altered CD31 expression

  • Decalcification of bone specimens can negatively impact CD31 antigenicity

How can CD31 antibodies be utilized in studies of leukocyte transendothelial migration?

CD31 plays a critical role in leukocyte transendothelial migration (TEM), making CD31 antibodies valuable tools for studying this process. Methodological approaches include:

• In vitro transmigration assays:

  • Endothelial monolayers grown on permeable supports (Transwell)

  • Pre-treatment of endothelial cells with inflammatory cytokines to upregulate adhesion molecules

  • Application of CD31 antibodies to block homophilic interactions or as detection reagents

  • Quantification of migrated leukocytes by flow cytometry or microscopy

• Live-cell imaging approaches:

  • Co-culture of fluorescently labeled leukocytes with endothelial monolayers

  • Real-time visualization of CD31 redistribution during TEM using fluorescently tagged CD31 antibodies

  • Analysis of CD31 clustering at the lateral border recycling compartment (LBRC)

• Mechanistic investigations:

  • Tyrosine-690 in CD31 plays a critical role in TEM and is required for efficient trafficking of PECAM1 to and from the LBRC

  • This residue is essential for targeting LBRC membrane around migrating leukocytes

  • CD31 heterophilic interaction with CD177 specifically mediates neutrophil transendothelial migration

• Validation controls:

  • Function-blocking CD31 antibodies to confirm CD31-dependent migration

  • Comparison with other adhesion molecule blockade (ICAM-1, VCAM-1)

  • siRNA knockdown of CD31 in endothelial cells as alternative to antibody blocking

What are the considerations for using CD31 antibodies in cancer research and angiogenesis studies?

CD31 antibodies are valuable tools in cancer research, particularly for studying tumor vasculature and angiogenesis. Key considerations include:

• Selection of appropriate antibody clone:

  • The JC70 clone has demonstrated superior capability for evaluating tumor angiogenesis and angiosarcoma compared to anti-FVIII-Reg antibodies

  • Recombinant versions offer improved consistency across experiments

• Quantification methodologies:

  • Microvessel density (MVD) assessment using standardized counting fields

  • Computer-assisted image analysis for more objective vessel quantification

  • 3D reconstruction techniques for volumetric assessment of vascular networks

• Prognostic correlations:

  • High levels of CD31 expression may indicate rapidly growing tumors

  • CD31 expression patterns can potentially predict tumor recurrence

  • Correlation with other angiogenic markers (VEGF, CD105) provides more comprehensive angiogenic profiling

• Tumor type considerations:

  • CD31 stains endothelial cells in both benign and malignant scenarios

  • Staining of non-vascular tumors (excluding hematopoietic neoplasms) is rare

  • Different tumor types exhibit varying vascular patterns requiring adjusted analysis parameters

• Therapeutic response assessment:

  • CD31 staining before and after anti-angiogenic therapy to assess vascular normalization

  • Combined with perfusion markers to distinguish functional from non-functional vessels

  • Integration with hypoxia markers to correlate vascular density with tissue oxygenation

How does CD31 antibody performance compare across different immunoassay platforms?

CD31 antibody performance varies across different platforms, with specific considerations for each application:

Western Blotting:

• Expected molecular weight: 130 kDa, though calculated MW is 83 kDa

• Discrepancy explained by post-translational modifications affecting protein mobility

• Sample preparation crucial: membrane enrichment improves detection

• Denaturation conditions can affect epitope accessibility

Immunohistochemistry:

• Paraffin-embedded samples require optimization of antigen retrieval

• Fresh-frozen tissues may preserve certain CD31 epitopes better than fixed tissues

• Signal amplification systems can enhance detection in tissues with low CD31 expression

• Chromogenic vs. fluorescent detection offers different advantages for co-localization studies

Flow Cytometry:

• Direct conjugated antibodies preferred to minimize background and non-specific binding

• Cell surface CD31 detection requires gentle cell preparation to preserve membrane integrity

• Intracellular CD31 detection necessitates appropriate permeabilization protocols

Multiplex Immunoassays:

• CD31 antibodies compatible with multiplex IHC/IF when appropriate fluorophores are selected

• Sequential staining may be necessary to avoid cross-reactivity in multiplex protocols

• Spectral unmixing techniques can improve signal discrimination in complex multiplex panels

Why might CD31 appear at different molecular weights in Western blot analysis?

The discrepancy between CD31's calculated molecular weight (83 kDa) and its observed molecular weight (130 kDa) in Western blot analysis stems from several factors:

• Post-translational modifications:

  • CD31 undergoes extensive glycosylation, significantly increasing its apparent molecular weight

  • Phosphorylation at multiple sites, particularly at ITIMs, alters protein mobility

  • Other modifications may include ubiquitination and proteolytic processing

• Sample preparation effects:

  • Incomplete denaturation can result in aggregates or retained secondary structure

  • Reduction conditions affect disulfide bonds that may influence protein migration

  • Sample buffer composition can impact SDS binding and thus apparent molecular weight

• Technical considerations:

  • Gel percentage and type significantly affect protein migration patterns

  • Running conditions (voltage, temperature) can influence band appearance

  • Molecular weight markers may not accurately predict migration of heavily modified proteins

As noted in the technical specifications, "the mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane" .

What storage and handling precautions ensure optimal CD31 antibody performance?

To maintain optimal CD31 antibody performance, researchers should adhere to these storage and handling guidelines:

• Storage temperature:

  • Store at -20°C for long-term preservation

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality

  • Aliquot antibodies upon receipt to minimize freeze-thaw events

• Shipping and temporary storage:

  • Shipment typically occurs with ice packs

  • Upon receipt, immediately transfer to recommended storage temperature

  • Short-term storage at 4°C (1-2 weeks) is generally acceptable for antibodies in use

• Buffer composition:

  • Typical formulation includes:

    • 50mM Tris-Glycine (pH 7.4)

    • 0.15M NaCl

    • 40% Glycerol

    • 0.05% stabilizer and protective protein

  • Buffer components help maintain antibody stability and prevent degradation

• Dilution practices:

  • Use high-quality, filtered buffers for dilutions

  • Add appropriate carriers (BSA, gelatin) to diluted antibodies to prevent adsorption to tubes

  • Prepare fresh dilutions for critical applications rather than storing diluted antibody

• Contamination prevention:

  • Use sterile technique when handling antibody vials

  • Include sodium azide (0.02-0.05%) in working dilutions to prevent microbial growth

  • Avoid repeated warming of stock vials to room temperature

What controls are essential when using CD31 antibodies in experimental designs?

Robust experimental design with CD31 antibodies requires implementation of appropriate controls:

• Positive controls:

  • Known CD31-expressing tissues/cells:

    • Human umbilical vein endothelial cells (HUVECs)

    • Human lung or kidney sections (rich in vessels)

    • Jurkat cells for Western blot applications

  • Verify antibody functioning under your specific experimental conditions

• Negative controls:

  • Primary antibody omission to assess secondary antibody specificity

  • Isotype-matched irrelevant antibodies to evaluate non-specific binding

  • CD31-negative cell lines (e.g., certain epithelial cell lines)

  • RNA interference or knockout models where CD31 expression is reduced/eliminated

• Technical controls:

  • Titration series to determine optimal antibody concentration

  • Multiple fixation/preparation methods comparison

  • Multiple detection systems assessment

  • Batch controls across experimental timepoints or conditions

• Validation approaches:

  • Confirmation with multiple CD31 antibody clones recognizing different epitopes

  • Correlation of protein detection with mRNA expression

  • Functional validation using blocking antibodies in biological assays

  • Cross-validation using complementary techniques (e.g., IF, flow cytometry, and WB)

How are CD31 antibodies being utilized in stem cell research?

CD31 antibodies have become increasingly valuable tools in stem cell research, with applications spanning identification, isolation, and functional characterization:

• Identification of hematopoietic stem cells:

  • CD31 is expressed by stem cells of the hematopoietic system

  • CD31 antibodies are used to identify and concentrate these cells for experimental studies and bone marrow transplantation

  • Flow cytometric analysis using CD31 as part of stem cell immunophenotyping panels

• Monitoring endothelial differentiation:

  • CD31 serves as a specific marker of endothelial differentiation

  • Antibodies enable tracking of stem cell differentiation toward endothelial lineages

  • Quantitative assessment of differentiation efficiency in various culture conditions

• Isolation of endothelial progenitors:

  • Magnetic or fluorescence-activated cell sorting using CD31 antibodies

  • Enrichment of vascular progenitors from heterogeneous stem cell populations

  • Purification of tissue-resident endothelial progenitors for regenerative medicine applications

• Functional vascular assessment:

  • Evaluation of vessel-forming capacity in stem cell-derived tissues

  • Characterization of vascular network complexity in organoids

  • Analysis of endothelial-perivascular cell interactions in tissue engineering

• Disease modeling:

  • Assessment of vascular phenotypes in patient-derived stem cell models

  • Drug screening using CD31 as a readout for vascular effects

  • Investigation of developmental vascular disorders using stem cell differentiation systems

What are the latest developments in CD31 antibody technology for advanced imaging applications?

Recent technological advances have expanded the capabilities of CD31 antibodies in advanced imaging applications:

• Super-resolution microscopy compatibility:

  • Direct conjugation with newer fluorophores optimized for STORM, PALM, or STED microscopy

  • Enhanced visualization of endothelial junctional complexes below the diffraction limit

  • Nanoscale distribution analysis of CD31 during leukocyte transendothelial migration

• Multiplexed imaging approaches:

  • Integration with cyclic immunofluorescence (CycIF) for highly multiplexed tissue analysis

  • Compatibility with mass cytometry imaging (IMC) for simultaneous detection of dozens of markers

  • Antibody conjugation with DNA barcodes for CODEX multiplexed imaging systems

• Intravital imaging applications:

  • Non-blocking fluorescently labeled CD31 antibodies for in vivo vascular visualization

  • Reduced antibody size formats (Fab fragments, nanobodies) for improved tissue penetration

  • Conjugation with near-infrared fluorophores for deeper tissue imaging

• 3D tissue imaging:

  • Optimization for whole-organ clearing techniques (CLARITY, iDISCO, CUBIC)

  • Long-term stability in clearing solutions and compatible mounting media

  • Software tools for automated vessel network reconstruction and analysis

• Functional imaging correlates:

  • Combined use with perfusion dyes to assess vessel functionality

  • Integration with hypoxia sensors to correlate vessel patterns with oxygen delivery

  • Correlation with mechanical sensors to study endothelial mechanobiology