CD81 Antibody, Biotin conjugated

What is CD81 and why is it an important target for research using biotin-conjugated antibodies?

CD81 (also known as TAPA-1 or TSPAN28) is a 26 kDa member of the tetraspanin superfamily characterized by four conserved transmembrane regions. CD81 plays crucial roles in diverse cellular interactions in both immune and nervous systems and participates in cell fusion events . It is expressed on multiple cell types, including:

• T and B lymphocytes

• NK cells

• Dendritic cells

• Thymocytes

• Endothelial cells

• Fibroblasts

CD81 forms multimolecular complexes with other cell surface proteins—associating with CD19, CD21, CD20, CD37, CD53, and CD82 in B cells, and with CD4, CD8, and CD82 in T cells . These interactions make CD81 a critical molecule in:

• B cell adhesion via VLA-4 integrin

• Early T cell development

• Signal transduction in the immune system

• Serving as a receptor for hepatitis C virus (HCV)

Biotin-conjugated CD81 antibodies provide exceptional utility for researchers due to their ability to leverage the strong biotin-streptavidin interaction, enabling sensitive detection and isolation of CD81-expressing cells and extracellular vesicles.

What are the primary applications for biotin-conjugated CD81 antibodies in laboratory research?

Biotin-conjugated CD81 antibodies serve diverse research applications across multiple experimental platforms:

These antibodies are particularly valuable for exosome research, allowing researchers to detect, quantitate, and characterize CD81-expressing extracellular vesicles (EVs) . When used in combination with streptavidin-based conjugates or streptavidin-precoated surfaces, they retain strong binding reactivity of the ligand . For comprehensive EV detection across different cell types and secretion pathways, researchers can employ CD81 antibodies alongside other tetraspanin markers like CD9 and CD63 .

How do different clones of biotin-conjugated CD81 antibodies compare in terms of species reactivity and applications?

Several biotin-conjugated CD81 antibody clones are available, each with distinct characteristics that researchers should consider when selecting the appropriate reagent:

The choice between these clones should be guided by your experimental system, target species, and specific application requirements. For instance, when analyzing mouse samples, the Eat-2 clone would be appropriate, whereas the 9B clone is better suited for human samples in ELISA applications.

How can I optimize the detection of exosomes using biotin-conjugated CD81 antibodies in ELISA-based assays?

For optimal exosome detection using biotin-conjugated CD81 antibodies in ELISA, consider the following methodological approach:

• Assay Setup: Implement a sandwich ELISA format using:

  • Capture antibody: Anti-tetraspanin (e.g., PS Capture™ platform)

  • Detection antibody: Biotin-conjugated anti-CD81

  • Secondary detection: Streptavidin-HRP conjugate

• Sample Preparation:

  • Human serum samples: Use direct detection without isolation

  • Cell culture supernatants: Consider concentration step for lower abundance EVs

  • Dilution series: Establish standard curves using purified exosomes (e.g., from COLO201 cells)

• Optimization Parameters:

  • Antibody concentration: For clone 9B, use 250 ng/mL for optimal signal-to-noise ratio

  • Determine appropriate working dilution (typically 1:2,000–1:16,000)

  • Block with appropriate buffers to reduce background signal

• Validation Controls:

  • Include isotype-matched control antibodies

  • Use exosome-depleted samples as negative controls

  • Employ purified exosomes as positive controls

Research data demonstrate that optimized biotin-conjugated anti-CD81 antibodies can achieve significantly lower blank values and higher EV signal values compared to competitor antibodies in ELISA systems . For comprehensive EV profiling, consider multiplexing with other tetraspanin markers (CD9, CD63) to detect exosomes of different cellular origins (endosomal vs. ectosomal) .

What factors should I consider when troubleshooting flow cytometry experiments using biotin-conjugated CD81 antibodies?

When troubleshooting flow cytometry experiments with biotin-conjugated CD81 antibodies, consider these key factors:

• Antibody Titration:

  • Optimize antibody concentration (recommended: ≤0.5 μg per 10^6 cells in 100 μL)

  • Perform titration experiments to determine optimal signal-to-noise ratio

  • Over-titration can lead to non-specific binding and high background

• Detection System:

  • Ensure appropriate streptavidin-fluorophore conjugate is used

  • Consider brightness of fluorophore relative to CD81 expression level

  • Account for potential spectral overlap in multi-color panels

• Cell Preparation:

  • Maintain cell viability above 90%

  • Ensure proper blocking to prevent Fc receptor binding

  • Consider fixation effects on epitope accessibility

• Experimental Controls:

  • Include isotype control (Mouse IgG1 for M38, Armenian Hamster IgG1 for Eat-2)

  • Use FMO (fluorescence minus one) controls for proper gating

  • Include positive control samples (e.g., thymocytes and splenocytes for Eat-2)

• Technical Considerations:

  • For mouse samples, Eat-2 shows reliable staining of CD81 on thymocytes and splenocytes

  • For human samples, clone M38 demonstrates consistent results with peripheral blood cells

  • Storage conditions: Keep at -20°C, avoid freeze-thaw cycles, and store in the dark

For difficult-to-detect populations, consider secondary amplification strategies or alternative formats of anti-CD81 antibodies with direct fluorophore conjugation for comparison of sensitivity.

How do biotin-conjugated CD81 antibodies perform in Western blotting applications compared to other formats?

When using biotin-conjugated CD81 antibodies for Western blotting, several technical considerations are crucial for optimal results:

• Sample Preparation:

  • Critical requirement: Use non-reducing conditions for CD81 detection

  • CD81 has a calculated molecular weight of 26 kDa but is typically observed at 22-25 kDa

  • Heat samples at lower temperatures (37°C) to preserve tetraspanin structure

• Optimized Protocol:

  • Recommended dilution: 1:1,000-1:8,000 for clone 9B

  • Transfer conditions: Use PVDF membrane with 0.2 μm pore size

  • Blocking: 5% non-fat milk or BSA to reduce background

  • Detection: Streptavidin-HRP followed by enhanced chemiluminescence

• Performance Comparison:

• Troubleshooting:

  • High background: Optimize blocking and washing steps

  • No signal: Confirm non-reducing conditions, verify protein loading

  • Multiple bands: May indicate post-translational modifications or proteolytic processing

Researchers should note that CD81's tetraspanin structure is dependent on proper disulfide bond formation, which is disrupted under reducing conditions, explaining why non-reducing conditions are essential for successful Western blot analysis .

How can biotin-conjugated CD81 antibodies be employed to investigate the role of CD81 in signal transduction pathways?

Biotin-conjugated CD81 antibodies serve as powerful tools for investigating CD81-mediated signal transduction through several methodological approaches:

• Immunoprecipitation of Signaling Complexes:

  • Use 5-20 μg of biotin-conjugated CD81 antibody per assay

  • Capture with streptavidin-coated beads or matrices

  • Analyze co-precipitated proteins by mass spectrometry

  • Research has identified ezrin as a key downstream component in CD81 signaling

• Investigation of Phosphorylation Events:

  • CD81 engagement induces specific tyrosine phosphorylation cascades

  • Key findings: CD81 ligation activates Syk kinase and induces ezrin phosphorylation at Y353

  • Sequential phosphorylation pattern:

    1. CD81 engagement → Syk autophosphorylation at Y525/526

    2. Activated Syk → Ezrin phosphorylation at Y353

    3. Ezrin phosphorylation → Actin cytoskeleton reorganization

• Cell-Type Specific Signaling Analysis:

  Cell Type	Primary Signaling Pathways	Key Molecules Activated
  B lymphocytes	Tyrosine phosphorylation cascades	Syk, Ezrin
  T cells	Src family kinase pathway	Lck
  Hepatocytes	MAPK-ERK and Rho GTPase	ERK, Rho GTPases

• Functional Assays:

  • CD81-induced ezrin phosphorylation leads to colocalization of CD81, ezrin, and F-actin

  • Syk inhibition (using R406) prevents this colocalization

  • Mechanistic explanation: CD81's C-terminal tail directly interacts with ezrin's N-terminal domain

These methodologies provide researchers with a comprehensive toolkit to dissect the molecular mechanisms by which CD81 influences diverse cellular processes including adhesion, morphology, activation, proliferation, and differentiation across different cell types.

What is the optimal methodology for using biotin-conjugated CD81 antibodies to isolate and characterize extracellular vesicles?

For rigorous isolation and characterization of extracellular vesicles (EVs) using biotin-conjugated CD81 antibodies, a systematic approach is recommended:

• Isolation Strategy:

  • Immunoaffinity Capture:

    • Use biotin-conjugated CD81 antibodies with streptavidin-coupled Dynabeads

    • Alternative: MSIA Streptavidin D.A.R.T.S can be employed

    • Advantage: Maintains native EV structure compared to ultracentrifugation

  • Differential Marker Approach:

    • Combine CD81 with other tetraspanin markers (CD9, CD63) for comprehensive isolation

    • Enables capture of both endosomal (CD63 enriched, CD9+, CD81+) and ectosomal (CD63 low, CD9+, CD81+) EVs

• Characterization Methodology:

  Method	Technical Approach	Data Output
  Sandwich ELISA	PS Capture™ platform with biotin-anti-CD81 (250 ng/mL)	Quantitative measurement of CD81+ EVs
  SPR/BLI	Immobilized biotin-CD81 antibody on streptavidin chip	Label-free detection, binding kinetics
  Flow Cytometry	Bead-based capture with secondary detection	Size and marker profile analysis

• Validation Controls and Parameters:

  • Use defined EV standards (e.g., Exosomes from COLO201 cells)

  • Include appropriate isotype controls

  • Verify purity by electron microscopy or nanoparticle tracking analysis

  • Confirm presence of additional EV markers (Alix, TSG101)

• Direct Detection in Biofluids:

  • Biotin-conjugated CD81 antibodies (particularly clone 9B) demonstrate superior performance for direct detection of EVs in human serum and EDTA plasma without isolation steps

  • Lower blank values and higher signal-to-noise ratios compared to competitor antibodies

This integrated approach leverages the specificity of biotin-conjugated CD81 antibodies while addressing the heterogeneity of EV populations, enabling researchers to conduct comprehensive EV analysis from isolation through detailed characterization.

How does CD81 engagement by antibodies affect ezrin phosphorylation and cytoskeletal reorganization in immune cells?

CD81 engagement by antibodies triggers a sophisticated signaling cascade that leads to ezrin phosphorylation and cytoskeletal reorganization:

• Molecular Signaling Pathway:

  • CD81 engagement → Syk activation (autophosphorylation at Y525/526)

  • Activated Syk → Ezrin phosphorylation at Y353

  • Ezrin phosphorylation → Altered ezrin conformation and F-actin binding

• Mechanistic Details:

  • Direct interaction occurs between CD81's C-terminal tail and ezrin's N-terminal domain

  • Ezrin contains an ITAM motif that enables interaction with Syk

  • Tyrosine phosphorylation of ezrin is associated with its redistribution to the plasma membrane

• Cytoskeletal Reorganization:

  • Following CD81 engagement, three key molecules colocalize at a single pole of stimulated cells:

    • CD81

    • Ezrin

    • F-actin

  • This polarization is Syk-dependent, as Syk inhibitor R406 prevents colocalization

• Functional Consequences:

  • Different outcomes in various immune cell types:

  Cell Type	Cytoskeletal Effect	Functional Outcome
  B cells	Cell polarization	Homotypic adhesion, early apoptotic events
  T cells	Enhanced motility	Increased activation when co-engaged with CD3
  NK cells	Altered cytoskeleton	Inhibited activation when co-engaged with CD16

• Experimental Validation:

  • Immunofluorescence microscopy confirms CD81/ezrin/F-actin colocalization

  • Biochemical analysis shows time-dependent increase in tyrosine-phosphorylated ezrin in CD81-stimulated cells

  • Both anti-CD81 mAbs and HCV envelope protein E2 induce this phosphorylation

This pathway represents a fundamental mechanism by which CD81 influences cell adhesion, morphology, and migration across diverse cell types, with significant implications for immune function and potential therapeutic targeting.

What approaches can be used to differentiate between CD81-positive EVs from different cellular origins using biotin-conjugated antibodies?

Distinguishing CD81-positive extracellular vesicles (EVs) from different cellular origins requires sophisticated analytical approaches using biotin-conjugated antibodies:

• Multi-marker Profiling Strategy:

  • Endosomal Origin EVs: CD63-enriched, CD9+, CD81+

  • Ectosomal Origin EVs: CD63-low, CD9+, CD81+

  • Implement parallel detection systems using:

    • CaptureSelect Biotin anti-CD81 conjugates

    • CaptureSelect Biotin anti-CD63 conjugates

    • CaptureSelect Biotin anti-CD9 conjugates

• Advanced Isolation Techniques:

  • Sequential Immunoaffinity:

    • First capture with one tetraspanin marker

    • Release and re-capture with a different marker

    • Creates subpopulation enrichment based on marker combinations

  • Density Gradient + Immunocapture:

    • Separate EVs by density characteristics first

    • Then apply biotin-CD81 capture to specific fractions

    • Enhances resolution of EV subpopulations

• Characterization Methods:

  Method	Technical Parameters	Differential Markers
  Multiplexed ELISA	Use PS Capture™ platform with different biotin-antibodies	CD81, CD9, CD63, cell-specific markers
  Flow Cytometry	Bead-based multi-parameter analysis	Tetraspanins + tissue-specific markers
  Mass Spectrometry	Protein profiling of immunocaptured EVs	Proteome signatures

• Biological Fluid Analysis:

  • Direct detection in biofluids using sandwich ELISA shows:

    • Different CD81/CD9/CD63 ratios between serum and plasma samples

    • Tissue-specific markers co-expression varies between sample types

    • Disease states alter tetraspanin expression patterns

• Data Interpretation Framework:

  • Create ratio analyses between different tetraspanin markers

  • Establish reference ranges for normal profiles in different biofluids

  • Compare unknown samples to established profiles for origin determination

This multi-dimensional approach leverages the specificity of biotin-conjugated CD81 antibodies while integrating additional markers to create a comprehensive EV fingerprinting system capable of distinguishing vesicles from diverse cellular sources.

What are the molecular mechanisms underlying CD81's diverse functions across different cell types?

CD81's diverse functions across cell types are governed by complex molecular mechanisms involving protein interactions, signaling pathways, and cellular context:

• Cell-Specific Protein Complex Formation:

  Cell Type	Key Interaction Partners	Functional Complex
  B cells	CD19, CD21, CD20, CD37, CD53, CD82	B-cell co-receptor complex
  T cells	CD4, CD8, CD82	T-cell signaling complex
  Hepatocytes	Claudin-1, SR-BI, EGFR	HCV entry complex
  Muscle cells	Unknown partners	Fusion machinery

• Divergent Signaling Pathway Activation:

  • B lymphocytes: CD81 engagement triggers Syk activation → ezrin phosphorylation → cytoskeletal reorganization

  • T cells: CD81 activates Src family kinase Lck → costimulation and motility enhancement

  • Hepatocytes: CD81 engagement activates MAPK-ERK and Rho GTPase pathways

  • NK cells: CD81 co-engagement with CD16 inhibits activation

• Structural Mechanisms:

  • CD81 organizes membrane microdomains (tetraspanin-enriched microdomains or TEMs)

  • The four transmembrane domains create a specific three-dimensional structure

  • Disulfide bonds in extracellular domains are critical for function

  • C-terminal cytoplasmic tail mediates interactions with intracellular signaling molecules, including ezrin

• Regulated Expression and Localization:

  • CD81 is expressed on virtually all nucleated cells

  • Highest expression on germinal center B cells

  • T cell activation induces CD81 upregulation

  • Subcellular localization changes upon cellular activation

• Functional Consequences:

  • Regulates adhesion, morphology, activation, proliferation, and differentiation of lymphocytes

  • Promotes cell fusion and myotube maintenance in muscles

  • In B cells, can induce early apoptotic events

  • Serves as receptor for hepatitis C virus

The integration of these multiple mechanisms explains how this single tetraspanin family member can mediate diverse effects across different cellular contexts, highlighting CD81's role as a versatile organizer of membrane-associated molecular complexes and signaling platforms.

How can researchers validate the specificity of biotin-conjugated CD81 antibody binding in complex biological samples?

Validating the specificity of biotin-conjugated CD81 antibody binding in complex biological samples requires a multi-faceted approach:

• Genetic Validation Approaches:

  • Knockout/Knockdown Controls:

    • Use CD81-knockout cell lines as negative controls

    • Compare siRNA/shRNA-treated versus non-treated samples

    • CRISPR-Cas9 edited cells provide definitive validation

  • Overexpression Systems:

    • Parallel analysis of CD81-overexpressing versus wild-type cells

    • Dose-dependent signal correlation with expression level

• Molecular Competition Assays:

  • Pre-incubate samples with unconjugated anti-CD81 antibody

  • Test different clones targeting distinct epitopes

  • Use recombinant CD81 protein for competitive binding

• Cross-Reactivity Assessment:

  Species	Clone Reactivity	Notes
  Human	M38, 9B, CaptureSelect VHH	All show strong reactivity
  Mouse	Eat-2 (EAT2)	Strong reactivity
  Rat	Eat-2 (EAT2)	Cross-reactive
  Bovine	9B (slight)	Weak cross-reactivity
  Rabbit, Cat	M38	Cross-reactive

• Technical Validation Strategies:

  • Orthogonal Detection Methods:

    • Compare biotin-antibody results with direct-labeled alternatives

    • Parallel validation using mass spectrometry

    • Western blot confirmation (under non-reducing conditions)

  • Controls for Non-Specific Binding:

    • Include isotype-matched control antibodies

    • Test binding to CD81-negative cells/tissues

    • Include blocking steps to reduce non-specific interactions

• Application-Specific Validation:

  • For flow cytometry: Use FMO (fluorescence minus one) controls

  • For ELISA: Compare signal-to-noise ratios with competitor antibodies

  • For EV analysis: Verify co-expression with other tetraspanin markers

• Biochemical Verification:

  • Immunoprecipitation followed by mass spectrometry

  • Size-exclusion chromatography to confirm appropriate molecular weight

  • Epitope mapping to verify binding to correct domain

This comprehensive validation approach ensures that experimental observations truly reflect CD81-specific binding rather than artifacts, particularly critical when working with heterogeneous samples such as extracellular vesicles or complex biological fluids.