SEMA4B Antibody, Biotin conjugated

What is SEMA4B and what biological functions does it regulate?

SEMA4B is a transmembrane homodimer glycoprotein belonging to the Class 4 semaphorin family. It functions as an important mediator of movement and differentiation across multiple cell types, including immune, vascular, and nervous systems . Recent research has identified SEMA4B's role in cancer progression, particularly in lung adenocarcinoma (LUAD), where it is significantly upregulated compared to normal tissue .

What are the optimal storage conditions for biotin-conjugated SEMA4B antibodies?

Biotin-conjugated SEMA4B antibodies require specific storage conditions to maintain their functionality and prevent degradation:

• Store aliquoted antibody at -20°C

• Avoid repeated freeze/thaw cycles that can degrade antibody activity

• Protect from light exposure, as biotin conjugates are often light-sensitive

• The antibody is typically supplied in a buffer containing 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol for stability

For long-term storage, creating multiple small aliquots upon receipt is recommended to minimize freeze/thaw cycles. Proper storage ensures the maintenance of >95% purity and optimal activity for experimental applications .

What applications have been validated for biotin-conjugated SEMA4B antibodies?

The applications validated for biotin-conjugated SEMA4B antibodies include:

While the biotin-conjugated version has been specifically validated for ELISA applications, unconjugated SEMA4B antibodies have demonstrated utility in multiple applications:

Researchers should conduct preliminary validation experiments to determine optimal working conditions for their specific experimental systems when using biotin-conjugated versions for applications beyond ELISA .

What controls should be included when using biotin-conjugated SEMA4B antibodies?

When designing experiments with biotin-conjugated SEMA4B antibodies, include these essential controls:

• Negative Controls:

  • Isotype control (rabbit IgG-biotin conjugated) to assess non-specific binding

  • No-primary antibody control to evaluate secondary reagent specificity

  • Known SEMA4B-negative tissues/cells to confirm specificity

• Positive Controls:

  • Human colon cancer tissue (validated as positive for SEMA4B expression)

  • LUAD tissue samples (shown to have upregulated SEMA4B expression)

• Biotin-Specific Controls:

  • Streptavidin-only control to assess endogenous biotin

  • Biotin blocking kit when working with tissues containing high endogenous biotin levels

• Validation Controls:

  • SEMA4B knockdown samples to confirm antibody specificity

  • Comparison with alternative SEMA4B antibody clones to verify staining patterns

Implementing these controls ensures experimental rigor and facilitates accurate interpretation of results when investigating SEMA4B expression and function.

How can I optimize immunohistochemistry protocols using biotin-conjugated SEMA4B antibodies?

Optimizing IHC protocols for biotin-conjugated SEMA4B antibodies requires attention to several parameters:

Antigen Retrieval Optimization:

• Test both TE buffer pH 9.0 (recommended) and citrate buffer pH 6.0 as alternatives

• Evaluate different retrieval durations (10-30 minutes) and temperatures

Blocking Endogenous Biotin:

• Use commercial biotin/avidin blocking kits before antibody application, especially crucial with biotin-conjugated primary antibodies

• Block endogenous peroxidase with H₂O₂ if using HRP-streptavidin detection

Antibody Dilution and Incubation:

• Test a dilution series starting from 1:50-1:500

• Compare overnight incubation at 4°C versus 1-2 hours at room temperature

Detection System Selection:

• Use fluorescent streptavidin conjugates for multiplexing capabilities

• Select enzymatic streptavidin-HRP systems for chromogenic detection and long-term slide archiving

Signal Amplification:

• Consider tyramide signal amplification for low-abundance SEMA4B detection

• Test streptavidin-poly-HRP systems for enhanced sensitivity

Based on published IHC data, SEMA4B shows positive detection in human colon cancer tissue and LUAD samples , providing valuable positive control tissues for protocol optimization.

How does SEMA4B expression correlate with immune cell infiltration in tumor microenvironments?

SEMA4B expression shows significant correlations with immune cell infiltration in tumor microenvironments, particularly in lung adenocarcinoma:

Positive Correlations with Immunosuppressive Cells:

• SEMA4B expression positively correlates with myeloid-derived suppressor cell (MDSC) infiltration (R = 0.368, p<0.001)

• SEMA4B expression positively correlates with regulatory T cell (Treg) infiltration (R = 0.143, p<0.05)

Impact on Cytotoxic Immune Cells:

• SEMA4B expression correlates with decreased infiltration of CD8+ T cells in LUAD, suggesting impaired anti-tumor immunity

Experimental Validation:

• Xenograft models with SEMA4B knockdown demonstrated decreased infiltration of T-regs and MDSCs in the tumor microenvironment

• Immunohistochemistry staining showed increased CD11b+ (MDSC marker) and Foxp3+ (Treg marker) cells in SEMA4B-positive LUAD samples compared to SEMA4B-negative samples

These findings suggest SEMA4B may mediate immune evasion by increasing recruitment of immunosuppressive cells, potentially explaining its association with poor prognosis in LUAD patients. Biotin-conjugated SEMA4B antibodies could be valuable tools for further investigating these immune infiltration patterns through multiplex immunohistochemistry approaches.

What approaches can be used to investigate SEMA4B's role in tumor proliferation and invasion?

Several methodological approaches can effectively investigate SEMA4B's role in tumor proliferation and invasion:

In Vitro Proliferation Assays:

• CCK-8 assay and EdU incorporation assay have successfully demonstrated reduced proliferation following SEMA4B knockdown in lung cancer cells

• Colony formation assays showed decreased colony numbers after SEMA4B silencing

Gene Silencing Strategies:

• siRNA transfection targeting SEMA4B (verified by qPCR at 72h post-transfection)

• shRNA stable transfection for longer-term studies and in vivo models

In Vivo Tumor Models:

• Subcutaneous injection of 2×10⁶ shSEMA4B/shCtrl-expressing bioluminescent tumor cells

• Tumor growth monitoring using bioluminescence imaging system

• Tumor measurement and weight assessment after animal sacrifice

Molecular Pathway Analysis:

• Investigation of PI3K-dependent MMP9 activation as a mechanism of SEMA4B-mediated invasion in NSCLC

• Western blot analysis of downstream signaling molecules in the PI3K pathway

Invasion and Migration Assays:

• Transwell migration assays with or without Matrigel

• Wound healing assays to assess cell migration capacity

A comprehensive research approach would combine these methods, using biotin-conjugated SEMA4B antibodies for detecting and quantifying SEMA4B expression across different experimental conditions and correlating this with functional outcomes.

How can I design experiments to investigate SEMA4B's potential as a therapeutic target?

Designing experiments to evaluate SEMA4B as a therapeutic target requires a systematic approach:

Target Validation:

• Analyze SEMA4B expression across patient cohorts using tissue microarrays with biotin-conjugated SEMA4B antibodies

• Correlate expression with clinical outcomes, as SEMA4B upregulation has been associated with later pathological stages and poor prognosis in LUAD patients

• Perform multivariate analysis to determine if SEMA4B is an independent prognostic factor

Mechanism of Action Studies:

• Investigate how SEMA4B modulates immune cell infiltration

• Examine the PI3K-dependent suppression of MMP9 activation pathway

• Study SEMA4B's interaction with its receptors through binding assays

Therapeutic Neutralization:

• Reference VX15/2503 approach, a humanized IgG4 monoclonal antibody developed against SEMA4D (another semaphorin family member)

• Design blocking antibodies against SEMA4B and evaluate their efficacy in preventing SEMA4B-receptor interactions

• Test antibody-mediated SEMA4B neutralization in animal models of lung cancer

Combination Therapy Assessment:

• Evaluate SEMA4B targeting in combination with immune checkpoint inhibitors

• Investigate synergistic effects with standard chemotherapy

• Test combination with targeted therapies against complementary pathways

Translational Models:

• Utilize patient-derived xenografts to evaluate SEMA4B targeting in models that better recapitulate tumor heterogeneity

• Develop organoid models incorporating immune components to study SEMA4B's immunomodulatory functions

These experimental approaches can help establish whether SEMA4B represents a viable therapeutic target, particularly in cancers like LUAD where it appears to promote tumor progression and immunosuppressive microenvironments.

How can I minimize background when using biotin-conjugated antibodies in tissues with high endogenous biotin?

Tissues with high endogenous biotin (like liver, kidney, and breast) present significant challenges when using biotin-conjugated antibodies. To minimize background:

Pre-analytical Solutions:

• Implement a dedicated biotin blocking step using commercial avidin/biotin blocking kits before antibody application

• Consider using fresh frozen tissues rather than FFPE when possible, as formalin fixation can sometimes expose more endogenous biotin

• Test alternative fixatives that may preserve antigenicity while minimizing biotin exposure

Analytical Approaches:

• Reduce primary antibody concentration (starting with 1:500 dilution and titrating as needed)

• Shorten incubation time with streptavidin detection reagents

• Include 0.1% BSA in washing buffers to reduce non-specific binding

Alternative Detection Strategies:

• Consider using non-biotin polymer detection systems if background persists

• Employ fluorescent secondary antibodies directly against rabbit IgG instead of biotin-streptavidin systems

• Test tyramide signal amplification methods which can allow for very low primary antibody concentrations

Control Experiments:

• Always include a streptavidin-only control (no primary antibody) to assess endogenous biotin levels

• Use isotype control antibodies (rabbit IgG-biotin) to evaluate non-specific binding

By implementing these strategies, researchers can achieve cleaner staining patterns when using biotin-conjugated SEMA4B antibodies, even in tissues with naturally high biotin content.

What are the best approaches for multiplexing studies involving biotin-conjugated SEMA4B antibodies?

Multiplexing with biotin-conjugated SEMA4B antibodies requires careful planning and optimization:

Sequential Multiplexing:

• Implement tyramide signal amplification (TSA) which allows antibody stripping between rounds

• Use biotin-conjugated SEMA4B antibody in the first round followed by complete stripping before subsequent rounds

• Validate complete stripping using no-secondary controls between rounds

Spectral Unmixing Approaches:

• Utilize spectral imaging systems capable of distinguishing multiple fluorophores

• Combine biotin-conjugated SEMA4B detection with directly-labeled antibodies against other targets

• Create comprehensive controls for spectral bleed-through

Multi-epitope-ligand cartography (MELC):

• Sequential imaging with photobleaching between cycles

• Well-suited for comprehensive immune cell profiling alongside SEMA4B

Recommended Target Combinations:
Based on SEMA4B's reported associations with immune infiltration , consider these multiplexing targets:

Validation Controls:

• Include single-stained controls for each fluorophore

• Run fluorescence-minus-one (FMO) controls

• Test potential cross-reactivity between detection systems

These approaches enable comprehensive spatial analysis of SEMA4B expression in relation to immune cell populations and other markers of interest in complex tissue microenvironments.

How do I interpret contradictory findings regarding SEMA4B's role in cancer progression?

The literature contains seemingly contradictory findings regarding SEMA4B's role in cancer:

Pro-tumor Evidence:

• SEMA4B expression is upregulated in LUAD tissues compared to normal tissue

• Higher SEMA4B expression correlates with later pathological stages and poor prognosis

• SEMA4B silencing suppresses lung cancer cell proliferation both in vitro and in vivo

• SEMA4B expression correlates with increased immunosuppressive cell infiltration (MDSCs, Tregs)

Anti-tumor Evidence:

• SEMA4B inhibits invasion of non-small cell lung cancer through PI3K-dependent suppression of MMP9 activation

To reconcile these findings:

Context-Dependent Functions:

• SEMA4B may have different roles in tumor initiation versus progression

• Effects may vary by cancer type, stage, or molecular subtype

• Receptor expression patterns in different tissues may dictate outcomes

Methodological Considerations:

• Knockdown versus overexpression studies may reveal different aspects of function

• In vitro versus in vivo studies may reflect microenvironmental influences

• Global versus cell-type-specific manipulation may obscure cell-autonomous effects

Research Design Approach:

• Design experiments that examine both proliferation and invasion/metastasis

• Include timing variables to assess stage-specific effects

• Consider receptor expression and signaling pathway activation states

These context-dependent functions are not uncommon for semaphorins, which often display pleiotropic effects depending on cellular context and receptor availability.

What can we learn from comparing SEMA4B to other semaphorin family members in therapeutic development?

Comparing SEMA4B to other semaphorins provides valuable insights for therapeutic development:

SEMA4D as a Precedent:

• VX15/2503, a humanized IgG4 monoclonal antibody against SEMA4D, has been developed for clinical applications

• VX15/2503 has entered clinical development for various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease

• The successful generation of SEMA4D antibodies in SEMA4D-deficient mice provides a methodological template for SEMA4B antibody development

Comparative Analysis Table:

Development Considerations:

• The epitope mapping and in vitro functional testing approaches used for VX15/2503 provide a roadmap for SEMA4B-targeted therapies

• Animal models used to demonstrate VX15/2503 efficacy in rheumatoid arthritis could inform SEMA4B therapeutic testing

• Biotin-conjugated antibodies may be valuable tools in preclinical studies before developing therapeutic-grade antibodies

This comparative analysis suggests that SEMA4B could follow a similar development trajectory to SEMA4D, with potential applications in both cancer and immune-mediated diseases based on its demonstrated roles in tumor progression and immune cell recruitment.