CD81 Monoclonal Antibody

What are the primary applications for CD81 monoclonal antibodies in research?

CD81 monoclonal antibodies are widely used in several laboratory techniques:

• Flow Cytometry: CD81 antibodies effectively detect CD81 expression on human lymphocytes and other cell types. These antibodies can be used with appropriate secondary antibodies, such as phycoerythrin-conjugated anti-mouse IgG, to visualize CD81-positive cells in flow cytometry applications .

• Western Blotting: Anti-CD81 antibodies are utilized to detect CD81 protein in cell lysates and extracellular vesicle (EV) preparations. Standard protocols typically employ 15-30 μg of protein lysate separated by SDS-PAGE followed by immunoblotting with the CD81 antibody .

• Immunocapture of Extracellular Vesicles: CD81 antibodies conjugated to beads can be used to isolate specific subpopulations of extracellular vesicles. This technique involves incubating antibody-conjugated beads with EVs isolated from cell culture media or biological fluids .

• Cell Migration Assays: CD81 antibodies can be employed to study the effects of CD81 neutralization on cell migration in various contexts, including immune cell trafficking and cancer cell motility .

How should CD81 antibodies be stored and handled for optimal performance?

For optimal performance, CD81 monoclonal antibodies should be:

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

• Avoid repeated freeze/thaw cycles which can diminish antibody activity

• Most CD81 antibodies are supplied in storage buffers containing stabilizers such as BSA, glycerol, and preservatives like Proclin300

• Working dilutions should be prepared fresh before use and stored at 4°C for short periods only

• Optimal dilution ranges vary by application: 1:300-1:5000 for Western blot applications

What controls should be included when using CD81 monoclonal antibodies?

Proper experimental controls are essential for interpreting results with CD81 antibodies:

• Isotype Controls: Use matched isotype control antibodies (such as mouse IgG1 for many CD81 mAbs) to determine non-specific binding .

• Positive Controls: Human lymphocytes or cell lines known to express CD81 (such as HMC-1.1 cells) .

• Negative Controls: When performing immunocapture experiments, unconjugated beads should be used as negative controls .

• Loading Controls: For Western blot experiments, appropriate loading controls should be included to normalize protein expression.

Epitope Specificity and Antibody Selection

When selecting a CD81 monoclonal antibody, researchers should consider:

• Experimental Application: Different clones may perform optimally in specific applications. For example, clone 454720 has been validated for flow cytometry applications , while clone 8C1 is recommended primarily for Western blot applications .

• Epitope Location: If studying specific domains of CD81, select antibodies that target relevant epitopes. The large extracellular loop contains most characterized epitopes, including those recognized by therapeutic antibodies .

• Functional Effects: For functional studies, consider whether cytokine induction is desirable or should be avoided. DSP-8250 lacks cytokine enhancement activity compared to 5A6, despite both inhibiting cell migration .

• Species Reactivity: Most characterized CD81 antibodies are specific to human CD81, though some may cross-react with other species .

How can CD81 monoclonal antibodies be utilized to study extracellular vesicle heterogeneity?

CD81 is a canonical marker of extracellular vesicles (EVs) and can be used to study EV subpopulations:

• Immunocapture Strategy: CD81 antibodies conjugated to beads can selectively capture CD81-positive EVs from heterogeneous populations. This approach has revealed distinct EV subpopulations with different marker profiles .

• Density Gradient Analysis: Following ultracentrifugation, CD81 antibodies can detect this marker in specific density fractions, helping characterize vesicle subpopulations. Research has shown that CD81 is particularly enriched in specific EV fractions (such as P120 fractions) compared to others .

• Differential Expression Analysis: Western blotting with CD81 antibodies alongside other tetraspanin markers (CD9, CD63) can reveal different EV subpopulations. Studies have demonstrated that EVs captured using CD81 antibodies show different marker profiles compared to those captured with CD9 or CD63 antibodies .

• Size and Concentration Measurement: When combined with techniques like nanoparticle tracking analysis, CD81 immunocapture can help determine the size distribution and concentration of specific EV subpopulations .

What mechanisms underlie the anti-metastatic effects of certain CD81 monoclonal antibodies?

Some CD81 monoclonal antibodies demonstrate inhibitory effects on cancer cell metastasis through several mechanisms:

• Inhibition of Cell Migration: Anti-CD81 antibodies can suppress cell migration in vitro and metastasis in vivo, particularly in breast cancer models .

• Protein Cross-linking: The anti-metastatic effects involve cross-linking CD81 molecules on the cell surface .

• Recruitment of Partner Proteins: Upon antibody binding, CD81 recruits specific partner proteins in the membrane, including:

  • Integrin β5

  • Integrin αV

  • Transferrin receptor 1 (TfR1)

• Domain-Specific Effects: The intracellular domains of CD81 are not essential for the anti-metastatic effects of the antibody, suggesting that the antibody's primary mechanism works through extracellular domain interactions and subsequent membrane protein organization .

How do CD81 antibodies differentially modulate immune cell functions?

CD81 antibodies can have diverse effects on immune functions, which are epitope-dependent:

• Migration Inhibition: Multiple CD81 antibodies (including 5A6 and DSP-8250) inhibit T-cell migration, which may be beneficial for treating inflammatory and autoimmune conditions .

• Cytokine Production: Some antibodies (like 5A6) increase cytokine production, which could exacerbate inflammatory conditions, while others (like DSP-8250) lack this activity despite retaining migration inhibition properties .

• Epitope Dependence: The functional differences between antibodies correlate with their epitope specificity. DSP-8250, which binds His151, Ala164, Ser168, and Asn172, uniquely inhibits migration without enhancing cytokines .

• Therapeutic Potential: Antibodies like DSP-8250 that separate beneficial (migration inhibition) from potentially harmful (cytokine induction) effects offer particular promise for therapeutic applications .

What are the optimal protocols for detecting CD81 in different cellular compartments?

Detection of CD81 requires different approaches depending on the cellular compartment:

• Cell Surface CD81:

  • Flow cytometry is the preferred method using live, non-permeabilized cells.

  • Typical protocol: Incubate cells with primary CD81 antibody (e.g., MAB4615) followed by a fluorochrome-conjugated secondary antibody .

  • For lymphocytes, whole blood samples can be directly stained, followed by red blood cell lysis.

• Total Cellular CD81:

  • Western blotting using whole cell lysates (30 μg protein) separated by SDS-PAGE.

  • Cell lysis buffers should contain appropriate detergents to solubilize membrane proteins .

• CD81 in Extracellular Vesicles:

  • Isolation methods include differential ultracentrifugation, density gradients, or size exclusion chromatography.

  • For Western blot, 15-30 μg of EV protein is typically used .

  • For immunocapture, antibody-conjugated beads are incubated with isolated EVs followed by washing and elution or direct analysis of bead-bound EVs .

How can researchers troubleshoot common issues with CD81 antibody applications?

Common challenges with CD81 antibody applications and their solutions include:

• Low Signal in Western Blot:

  • Increase antibody concentration (try 1:300 dilution)

  • Optimize protein loading (30 μg is recommended for cell lysates, 15 μg for EV preparations)

  • Ensure proper membrane transfer of this tetraspanin protein

  • Use enhanced chemiluminescence detection systems

• High Background in Flow Cytometry:

  • Include proper blocking steps with serum matching the secondary antibody host

  • Use isotype control antibodies to determine non-specific binding

  • Optimize antibody concentration through titration

  • Ensure cells are properly washed between steps

• Inconsistent EV Immunocapture:

  • Standardize starting EV concentration (e.g., 1.9 × 10^10 EVs)

  • Optimize antibody-to-bead conjugation procedures

  • Include unconjugated beads as negative controls

  • Standardize incubation times and washing procedures

How are CD81 monoclonal antibodies being employed in cancer research beyond detection methods?

CD81 monoclonal antibodies are finding novel applications in cancer research:

• Anti-Metastatic Therapeutics: Certain CD81 antibodies demonstrate inhibitory effects on breast cancer metastasis in xenograft models by binding to unique epitopes in the large extracellular loop .

• Molecular Mechanism Studies: CD81 antibodies have revealed that cross-linking of CD81 molecules and recruitment of partner proteins (integrin β5, integrin αV, and transferrin receptor 1) contribute to anti-metastatic effects .

• Functional Domain Analysis: Research using CD81 antibodies has determined that intracellular domains of CD81 are not essential for the anti-metastatic effects, focusing attention on extracellular interactions .

• EV Subpopulation Analysis: CD81 antibodies are being used to isolate and characterize cancer-derived EV subpopulations, which may have diagnostic or prognostic value .

What are the emerging applications of CD81 antibodies in immunotherapy research?

Recent research highlights several promising applications of CD81 antibodies in immunotherapy:

• Selective Immune Modulation: Antibodies like DSP-8250 can inhibit immune cell migration without enhancing cytokine production, potentially offering more selective immunomodulation than existing approaches .

• Epitope-Based Drug Design: The identification of specific epitopes (His151, Ala164, Ser168, and Asn172) that allow separation of beneficial and harmful effects provides targets for rational drug design .

• Combination Therapies: CD81 antibodies might complement existing immunotherapies by addressing specific aspects of immune cell trafficking without exacerbating inflammation through cytokine induction .

• Autoimmune Disease Treatment: The ability to inhibit leukocyte migration suggests potential applications in treating autoimmune conditions where pathological immune cell infiltration drives disease .

What are the current limitations of CD81 monoclonal antibodies in research applications?

Despite their utility, CD81 monoclonal antibodies face several limitations:

• Epitope Accessibility: The tetraspanin structure of CD81, with four transmembrane domains, can limit epitope accessibility in certain applications or fixation conditions.

• Functional Variability: Different antibody clones produce dramatically different functional effects, making standardization challenging .

• Species Cross-Reactivity: Many commercially available CD81 antibodies are specific to human CD81, limiting their use in animal models .

• Context-Dependent Effects: The effects of CD81 antibodies may vary depending on cell type, activation state, and the presence of CD81 partner proteins, complicating interpretation.

How might emerging technologies enhance CD81 antibody development and applications?

Future directions for CD81 antibody research may include:

• Single-Domain Antibodies: Development of nanobodies or single-domain antibodies against CD81 that might access epitopes unavailable to conventional antibodies.

• Bispecific Antibodies: Creation of bispecific antibodies targeting CD81 and partner proteins to enhance specificity for particular cellular contexts.

• Structure-Guided Engineering: With greater understanding of CD81's structure and epitope mapping, more precisely engineered antibodies with specific functional properties could be developed .

• High-Throughput Screening: Application of phage display and other high-throughput technologies to identify novel anti-CD81 antibodies with unique properties, similar to how DSP-8250 was discovered .

• In Vivo Imaging: Development of CD81 antibody-based imaging agents to track EV distribution or CD81-expressing cells in vivo.