CD81 Antibody

What is CD81 and why is it important in cellular research?

CD81 (also known by accession number P60033) is a tetraspanin protein expressed on the surface of numerous cell types, particularly lymphocytes, and serves as a canonical extracellular vesicle (EV) marker protein . The importance of CD81 in cellular research stems from its involvement in various biological processes including cell adhesion, motility, activation, and signal transduction. CD81 is widely distributed across human cells and tissues, making it a valuable target for studying cellular communication and membrane organization. This protein is frequently used as a marker for characterizing extracellular vesicles, which are membrane-enclosed structures released by cells that play crucial roles in intercellular communication . Additionally, CD81 functions as a receptor for Hepatitis C Virus (HCV), which makes it particularly relevant for virology research and the development of antiviral strategies .

How do antibodies against CD81 function in experimental systems?

CD81 antibodies function by specifically binding to epitopes on the CD81 protein, allowing for the detection, quantification, and functional analysis of this tetraspanin in various experimental systems. In flow cytometry applications, anti-CD81 antibodies can identify and enumerate CD81-expressing cells within heterogeneous populations, such as lymphocytes in whole blood samples . For Western blot applications, these antibodies recognize denatured CD81 protein separated by SDS-PAGE, enabling detection of CD81 in cell lysates and extracellular vesicle preparations . In immunocapture techniques, CD81 antibodies can be conjugated to solid supports like beads to isolate CD81-expressing EVs from complex biological fluids, facilitating downstream analysis of these vesicles . Furthermore, certain monoclonal anti-CD81 antibodies have been shown to functionally block CD81-dependent processes, such as HCV entry into hepatocytes, making them valuable tools for investigating the biological roles of CD81 and its potential as a therapeutic target .

What are the common applications of CD81 antibodies in basic research?

CD81 antibodies are employed across diverse applications in basic research, with flow cytometry being a predominant technique for analyzing CD81 expression on cell surfaces. Flow cytometric analysis using CD81 antibodies allows researchers to identify CD81-positive cells within complex mixtures like whole blood lymphocytes, providing insights into cell phenotyping and population dynamics . Western blotting represents another critical application, where anti-CD81 antibodies detect the protein in cell and extracellular vesicle lysates, enabling comparative expression analysis across different samples and experimental conditions . Immunocapture of extracellular vesicles using CD81 antibodies has become increasingly important for isolating and characterizing specific EV subpopulations, as demonstrated in studies where CD81-conjugated beads were used to capture EVs for subsequent proteomic analysis . Beyond these applications, CD81 antibodies are valuable for immunohistochemistry, immunoprecipitation, and functional blocking studies, particularly in viral infection models where anti-CD81 antibodies can inhibit HCV infection by targeting post-binding steps during viral entry .

What are the optimal techniques for detecting CD81 using antibodies?

The detection of CD81 using antibodies can be optimized through several complementary techniques, with flow cytometry offering exceptional sensitivity for cell surface expression analysis. For optimal flow cytometry results, researchers should use fluorophore-conjugated anti-CD81 antibodies or employ a primary anti-CD81 antibody followed by fluorophore-conjugated secondary antibodies, such as Phycoerythrin-conjugated Anti-Mouse IgG F(ab')2 . Western blotting provides a powerful approach for CD81 protein detection in cell and EV lysates, with optimal results achieved by loading 15-30 μg of protein, separating by SDS-PAGE, and probing with specific anti-CD81 antibodies followed by appropriate secondary antibodies . Immunocapture techniques using antibody-conjugated beads represent another valuable approach, particularly for isolating CD81-positive EVs from complex samples, as demonstrated in studies where different antibodies targeting CD81 were used to capture specific vesicle populations . Size exclusion chromatography (SEC) combined with immunoblotting offers an additional strategy for CD81 detection, allowing for the separation of EVs from soluble proteins prior to antibody-based detection, thus enhancing specificity and reducing background interference .

How can I optimize flow cytometry protocols for CD81 detection in lymphocytes?

Optimizing flow cytometry protocols for CD81 detection in lymphocytes requires careful attention to several key parameters including antibody concentration, incubation conditions, and appropriate controls. When working with human whole blood lymphocytes, researchers should first isolate the lymphocyte population through density gradient centrifugation or use whole blood lysis protocols to remove erythrocytes while preserving leukocytes . Titration of the primary anti-CD81 antibody (such as clone 454720) is essential to determine the optimal concentration that maximizes specific signal while minimizing background, with dilutions typically requiring empirical determination for each application . Incorporation of appropriate controls is crucial, including isotype control antibodies (such as MAB0041) to assess non-specific binding and unstained controls to establish autofluorescence baselines . When using indirect staining approaches, researchers should select appropriate secondary antibodies, such as Phycoerythrin-conjugated Anti-Mouse IgG F(ab')2 (Catalog # F0102B), ensuring they are compatible with the primary antibody isotype and optimized for minimal cross-reactivity . Sample processing time should be minimized to preserve cell viability and surface antigen integrity, with staining performed at 4°C to reduce potential internalization of CD81 during the procedure .

What are the best practices for Western blot detection of CD81 in different sample types?

Western blot detection of CD81 across various sample types requires specific optimization strategies to address the unique challenges presented by different biological materials. For cellular samples, optimal protein loading typically ranges from 20-30 μg per lane, while EV samples may require 15-30 μg depending on CD81 abundance, with all samples properly denatured in SDS-containing buffer before electrophoresis . When analyzing extracellular vesicle fractions, researchers should include appropriate cellular controls and exclusion markers like calreticulin to confirm EV purity and distinguish EVs from cellular contaminants . The selection of antibodies requires careful consideration, with monoclonal antibodies offering high specificity for CD81 detection, though the epitope accessibility may differ between native and denatured protein forms . For samples from density gradient fractionation experiments, normalization strategies should be implemented to account for variation in vesicle concentration across fractions, with protein loading adjusted based on particle numbers determined by nanoparticle tracking analysis . Researchers should be mindful that CD81 detection patterns may vary between different EV subpopulations (such as P15 versus P120 fractions) due to heterogeneous distribution, necessitating comparative analysis across multiple EV isolation methods to comprehensively characterize CD81 expression .

How can I effectively use CD81 antibodies for extracellular vesicle immunocapture?

Effective immunocapture of extracellular vesicles using CD81 antibodies requires systematic optimization of multiple parameters to ensure high specificity and yield. The selection of appropriate antibodies is crucial, with those targeting accessible epitopes on the native CD81 protein proving most effective for capturing intact EVs, as demonstrated in studies using different antibodies against N-terminal, C-terminal, or full-length CD81 . Antibody coupling to solid supports, typically magnetic beads, should follow established conjugation protocols with validation of coupling efficiency through protein quantification or functional binding assays . The ratio of EVs to antibody-conjugated beads requires empirical optimization, with studies showing successful immunocapture using 1.9-3.5 × 10^10 EVs per capture reaction, though this may vary depending on CD81 expression levels and bead capacity . Incubation conditions significantly impact capture efficiency, with optimal results typically achieved using gentle rotation at 4°C for several hours to overnight, balancing binding kinetics with vesicle integrity preservation . After capture, elution methods must be carefully selected based on downstream applications, with mild elution preserving EV integrity for functional studies versus stronger elution conditions for comprehensive protein analysis by Western blotting or mass spectrometry .

How are CD81 antibodies utilized in studying extracellular vesicle heterogeneity?

CD81 antibodies serve as powerful tools for investigating extracellular vesicle heterogeneity by enabling the isolation and characterization of distinct EV subpopulations with varying molecular compositions. Comparative immunocapture studies using antibodies against CD81 alongside other tetraspanins such as CD9 and CD63 have revealed the existence of distinct EV subsets with different protein compositions, suggesting functional specialization among vesicle populations . Differential ultracentrifugation combined with CD81 immunoblotting has demonstrated that CD81 is differentially distributed between various EV fractions, with studies showing enrichment of CD81 in smaller P120 EV fractions compared to larger P15 fractions, highlighting size-dependent heterogeneity in tetraspanin distribution . Density gradient fractionation followed by CD81 immunodetection provides further resolution of EV subpopulations, with CD81-positive vesicles predominantly found in specific density ranges, allowing for more precise isolation of functionally distinct EV subtypes . Advanced multiplexed approaches combining CD81 with other markers like KIT receptor have uncovered novel EV subpopulations with unique protein signatures, demonstrating how CD81 antibodies can be integrated into multi-parameter analyses to map the complex landscape of EV heterogeneity and inform studies of their diverse biological functions .

What is the role of CD81 in HCV infection and how do anti-CD81 antibodies block viral spread?

CD81 plays a critical role in Hepatitis C Virus (HCV) infection by serving as an essential entry factor that facilitates viral binding and internalization into host cells. Novel anti-CD81 monoclonal antibodies, particularly those generated through genetic immunization, have demonstrated remarkable efficacy in inhibiting HCV infection across different viral genotypes, including escape variants selected during liver transplantation that re-infected liver grafts . Mechanistic studies of these antibodies have revealed that they primarily target post-binding steps during HCV entry rather than initial attachment, suggesting they disrupt critical CD81-dependent conformational changes or interactions required for successful viral internalization . Beyond their ability to block cell-free HCV infection, certain anti-CD81 monoclonal antibodies have shown the capacity to inhibit neutralizing antibody-resistant HCV cell-cell transmission, a key pathway for viral dissemination within the liver that typically evades humoral immune responses . This dual inhibitory activity against both cell-free infection and cell-cell spread represents a significant advantage for research applications and potential therapeutic development, as it addresses multiple routes of viral propagation without displaying detectable toxicity toward host cells . The ability of these antibodies to efficiently block viral dissemination makes them valuable tools for further unraveling the complex virus-host interactions during HCV entry and cell-cell transmission, potentially informing the development of novel antiviral strategies .

How can researchers distinguish between different EV subpopulations using CD81 and other markers?

Researchers can effectively distinguish between different extracellular vesicle subpopulations by implementing multi-parameter analysis strategies that combine CD81 with other EV markers and physical separation techniques. Sequential immunocapture approaches using antibodies against CD81 followed by analysis of additional markers allow researchers to identify distinct vesicle subsets, as demonstrated in studies where CD81-captured EVs showed different marker profiles compared to CD9-enriched vesicles, with specific enrichment patterns for proteins like flotillin-1 and KIT . Differential ultracentrifugation protocols separating vesicles into P15 (larger) and P120 (smaller) fractions, followed by CD81 immunoblotting, reveal size-dependent heterogeneity in EV populations, with CD81 often showing preferential association with smaller vesicle fractions while other markers like annexin A1 may predominate in larger fractions . Density gradient fractionation combined with CD81 detection enables further resolution of EV subpopulations based on their buoyant density, with immunoblot analysis revealing that CD81-positive vesicles often distribute across specific density ranges (typically reported in g/ml), allowing for isolation of functionally distinct EV subtypes . Advanced multiplexed approaches using antibodies against CD81 along with other tetraspanins (CD9, CD63) and cell-specific markers like KIT receptor can uncover novel EV subsets with unique molecular signatures, as shown in comparative immunoblotting of captured fractions where distinct protein enrichment patterns were observed across different EV subpopulations .

What methodologies exist for evaluating CD81 antibody specificity and functionality?

Evaluating CD81 antibody specificity and functionality requires rigorous validation through complementary methodologies that assess both binding characteristics and biological activity. Flow cytometry represents a primary approach for specificity assessment, comparing staining patterns between target cells known to express CD81 (such as lymphocytes) and appropriate controls, including isotype antibodies and CD81-negative or knockdown cells . Western blot validation should include positive controls (CD81-expressing cells/EVs), negative controls, and evaluation of band specificity at the expected molecular weight, with additional validation through comparative analysis using multiple anti-CD81 antibodies targeting different epitopes . Immunocapture experiments coupled with downstream analysis can assess functional binding to native CD81, with successful vesicle isolation confirmed by detecting multiple EV markers in captured fractions and appropriate depletion in flow-through samples . For antibodies intended for functional blocking applications, biological activity assays such as HCV infection inhibition provide critical validation, with effective antibodies demonstrating concentration-dependent blockade of viral infection and cell-cell spread without cellular toxicity . Cross-reactivity assessment across species and cell types is essential for determining application scope, while epitope mapping through techniques like peptide arrays or competition assays provides valuable information about the antibody's binding site and potential for functional interference with CD81-dependent processes .

Why might researchers observe different CD81 expression levels in various EV fractions?

Differential CD81 expression across extracellular vesicle fractions stems from multiple biological and methodological factors that reflect the inherent heterogeneity of these membrane-enclosed structures. The biogenesis pathway significantly influences CD81 incorporation, with vesicles originating from multivesicular bodies (typically found in P120 fractions) often showing higher CD81 content compared to those derived from direct plasma membrane budding (more common in P15 fractions), as demonstrated in studies where CD81 was predominantly detected in P120 EV fractions while annexin A1 was enriched in P15 fractions . Physical properties of EVs, including size and density, correlate with differential protein composition, with smaller vesicles (50-150 nm) typically showing higher CD81 expression than larger vesicles (>200 nm), an observation supported by nanoparticle tracking analysis of different EV subpopulations . Cell type-specific factors significantly impact CD81 distribution, as exemplified by studies comparing EVs derived from different cell lines like HMC-1.1 and HMC-1.2, where distinct CD81 expression patterns were observed despite similar isolation methods . Additionally, isolation methodology introduces significant variability, with different approaches (ultracentrifugation, density gradients, size exclusion chromatography, immunoaffinity capture) each exhibiting selective bias toward specific EV subpopulations with varying CD81 content, necessitating method standardization and comparative analysis for accurate interpretation .

How can researchers troubleshoot inconsistent CD81 detection in Western blot experiments?

Troubleshooting inconsistent CD81 detection in Western blot experiments requires systematic evaluation of multiple technical parameters affecting protein extraction, separation, transfer, and immunodetection. Sample preparation techniques critically impact CD81 detection, with extraction buffers containing appropriate detergents (RIPA or NP-40) needed to effectively solubilize this transmembrane protein from lipid-rich membranes, while maintaining consistent sample handling conditions (temperature, processing time) to prevent protein degradation . Electrophoresis conditions, including protein loading amount (optimally 15-30 μg), gel percentage (typically 10-12% for CD81), and running parameters, must be optimized to achieve proper protein separation and transfer efficiency, with oversaturated or underloaded samples leading to inconsistent detection . Transfer efficiency greatly influences detection consistency, with semi-dry and wet transfer protocols requiring optimization for hydrophobic membrane proteins like CD81, potentially benefiting from extended transfer times or modified buffer compositions containing methanol to facilitate transfer of lipid-associated proteins . Antibody selection and optimization represent critical factors, with monoclonal antibodies offering high specificity but potentially limited epitope accessibility in denatured proteins, requiring comparison of multiple antibodies targeting different CD81 regions, as demonstrated in studies using antibodies against N-terminal, C-terminal, and full-length CD81 . Post-transfer membrane handling significantly impacts detection, with optimal blocking conditions (typically 5% non-fat milk or BSA), antibody dilutions, incubation times/temperatures, and washing protocols requiring empirical determination to maximize signal-to-noise ratio and ensure reproducible CD81 detection across experiments .

How do researchers interpret contradictory results between different CD81 detection methods?

Interpreting contradictory results between different CD81 detection methods requires understanding the fundamental differences in sample preparation, detection principles, and sensitivity limits of each technique. Epitope accessibility variations significantly impact detection across methods, with flow cytometry detecting native CD81 in its membrane environment while Western blotting detects denatured protein, potentially leading to discrepancies when antibodies recognize conformation-dependent epitopes, as illustrated in studies using antibodies targeting different CD81 regions with varying detection efficiencies across methods . Sample preparation differences introduce methodological biases, with flow cytometry analyzing intact vesicles or cells, while Western blotting examines lysed samples, potentially affecting CD81 detection through differential protein extraction efficiency or epitope masking during processing . Sensitivity thresholds vary substantially between methods, with flow cytometry offering superior sensitivity for detecting low-abundance CD81 on individual vesicles or cells, while Western blotting requires larger vesicle populations to generate detectable signals, resulting in potential detection in one method but not the other depending on expression levels . Heterogeneity in EV populations presents a major source of apparent contradictions, with bulk analyses potentially obscuring subpopulation-specific characteristics, as demonstrated in studies where distinct EV fractions showed dramatically different CD81 expression profiles despite originating from the same cells . Resolution of contradictory results typically requires triangulation using additional methods or markers, with immunoelectron microscopy, mass spectrometry-based proteomics, or super-resolution microscopy providing orthogonal validation and helping reconcile apparent discrepancies between conventional detection approaches .