Sema3a Antibody

What is Sema3A and what cellular functions does it regulate?

Semaphorin 3A (Sema3A) is a secreted protein belonging to the semaphorin family that functions primarily as a chemorepellent in neural development. It plays a crucial role in axonal guidance by directing the growth of neural axons to form intricate neural networks . Beyond its neurological functions, Sema3A is involved in regulating angiogenesis through its interaction with neuropilin-1 (NP-1) and neuropilin-2 receptors, where it competes with vascular endothelial growth factor (VEGF) and suppresses new blood vessel formation . This competitive binding mechanism has linked Sema3A to cancer progression and other pathological conditions including glioblastoma (GBM) . Sema3A is also expressed on regulatory B cells (CD19+CD25high) and has immunomodulatory properties that can influence autoimmune conditions such as systemic lupus erythematosus (SLE) .

What types of Sema3A antibodies are currently available for research applications?

Several types of Sema3A antibodies are available for research purposes, each with specific characteristics:

These antibodies vary in their specificity, cross-reactivity with species orthologs, and availability in conjugated forms (HRP, PE, FITC, Alexa Fluor conjugates) .

What are the validated methodological applications for Sema3A antibodies?

Sema3A antibodies have been validated for multiple experimental applications with specific methodological considerations:

• Western Blotting (WB): Typically used at dilutions of 1:500, can detect both the 95 kDa and 125 kDa forms of recombinant Sema3A . Recommended for analyzing tissue lysates and recombinant proteins.

• Immunoprecipitation (IP): Effective for isolating Sema3A protein complexes from cell lysates to study protein-protein interactions.

• Immunofluorescence (IF): Used to visualize Sema3A distribution in cultured cells, typically at concentrations of 1-5 μg/ml.

• Immunohistochemistry (IHC): Applied at approximately 1 μg/ml concentration on formalin-fixed, paraffin-embedded tissues after appropriate antigen retrieval (e.g., using EDTA pH 9.0) .

• ELISA: Generally employed at 1:2000 dilution for detecting Sema3A in serum samples, which has proven valuable for studying conditions like SLE .

• Functional Neutralization: Anti-Sema3A antibodies such as F11 can be used at concentrations of 10 μg/ml to block Sema3A function in cellular assays .

How should researchers interpret serum Sema3A levels in different pathological conditions?

Interpretation of serum Sema3A levels requires careful consideration of disease context and control selection:

In SLE patients, serum Sema3A levels are significantly lower (55.04 ± 16.30 ng/ml) compared to healthy controls (74.41 ± 17.60 ng/ml, P < 0.0001) and RA patients (65.54 ± 14.82 ng/ml, P = 0.018) . These reduced levels negatively correlate with:

• Disease activity (SLEDAI score)

• Renal damage

• Presence of anti-cardiolipin antibodies

• Complement C3-C4 serum levels

When analyzing Sema3A levels, researchers should:

• Include appropriate age and sex-matched controls

• Consider disease heterogeneity within patient populations

• Correlate with other disease markers for comprehensive interpretation

• Use consistent measurement techniques (typically ELISA with anti-Sema3A antibodies)

• Account for potential confounding factors such as medication use

These methodological considerations ensure reliable interpretation of Sema3A levels as potential biomarkers for disease activity, particularly in autoimmune conditions .

How can researchers optimize neutralizing anti-Sema3A antibodies for therapeutic development?

Development of therapeutically effective neutralizing anti-Sema3A antibodies involves several sophisticated optimization steps:

• Antibody Engineering: Using phage display technology with synthetic antibody libraries to isolate anti-Sema3A single-chain variable fragments (scFvs) with high binding affinity. This approach has successfully generated fully human IgG1 monoclonal antibodies such as F11, A08, and C10 clones .

• Affinity Maturation: Measuring KD values (equilibrium dissociation constant) using surface plasmon resonance assays to identify antibodies with the highest binding affinity. The F11 IgG antibody demonstrated superior binding affinity to both human and mouse Sema3A compared to other clones .

• Cross-Reactivity Testing: Validating antibody reactivity across multiple species (human, mouse, rat) to facilitate translational research. For example, the F11 antibody showed positive cross-reactivity with mouse Sema3A due to approximately 95% sequence homology .

• Functional Validation: Testing neutralizing capacity in cellular assays such as:

  • Migration assays in transwell chambers (using 10 μg/ml IgG)

  • Cytoskeletal collapse assays measuring cellular impedance

  • Endothelial cell permeability assays using FITC-coupled dextran

• Pharmacokinetic Analysis: Evaluating PK profiles through administration of single intravenous bolus doses (e.g., 5 or 25 mg/kg in BALB/c mice), which has shown biological half-life measurements of approximately 6.93 days at 5 mg/kg .

• In Vivo Efficacy Testing: Using appropriate disease models such as GBM patient-derived xenograft (PDX) models or oxygen-induced retinopathy models to evaluate therapeutic effects .

These optimization approaches have led to promising candidates like BI-X, an intravitreal anti-Sema3A agent currently under investigation for proliferative diabetic retinopathy and diabetic macular ischemia .

What are the key considerations for using anti-Sema3A antibodies in neurological research?

When utilizing anti-Sema3A antibodies for investigating neurological mechanisms, researchers should consider:

• Spatiotemporal Expression Analysis: Sema3A expression varies across development and brain regions. Immunohistochemical staining of cerebellum sections using anti-Sema3A antibodies (e.g., PAB7888) has revealed distinct expression patterns that must be considered when designing experiments .

• Receptor Complex Interactions: Sema3A functions through binding to the neuropilin-1/plexin-1 complex. Experimental designs should account for these interactions, potentially including co-immunoprecipitation studies to examine complex formation .

• Growth Cone Collapse Assays: Anti-Sema3A antibodies can be used to neutralize Sema3A-induced collapse and paralysis of neuronal growth cones. Quantifying growth cone morphology requires standardized imaging and analysis protocols .

• Neuronal Guidance Measurements: Transwell migration assays with neuronal cells treated with anti-Sema3A antibodies (scFv at 50 μg/ml or IgG at 10 μg/ml) provide quantitative data on migratory behavior .

• Specificity Controls: Given the homology between semaphorin family members, controls using related proteins (e.g., Sema3C, Sema3E, Sema3B, Sema3F) are essential to confirm antibody specificity .

• Developmental Studies: For research on olfactory system development or neuronal control of puberty, stage-specific application of anti-Sema3A antibodies is critical for accurate interpretation of results .

These methodological considerations enable researchers to effectively investigate Sema3A's role in neural development, axonal guidance, and related neurological processes.

How do researchers measure the efficacy of anti-Sema3A antibodies in angiogenesis-related studies?

Assessment of anti-Sema3A antibody efficacy in angiogenesis research requires specialized methodological approaches:

• Endothelial Cell Permeability Assays:

  • Human retinal microvascular endothelial cells (HRMECs) are treated with recombinant Sema3A (500 ng/ml) with or without anti-Sema3A antibodies (1 μg/ml)

  • Permeability is quantified by measuring the passage of FITC-coupled dextran through the endothelial cell layer

  • Controls include anti-TNP antibodies (1 μg/ml) and VEGF-A (100 ng/ml) treatments for comparison

• Cytoskeletal Collapse Measurements:

  • Cellular impedance reductions indicate cytoskeletal collapse

  • Concentration-response curves with recombinant human Sema3A determine potency

  • Specificity is verified by comparing effects of Sema3A vs. Sema3C, Sema3E, Sema3B, or Sema3F

• Oxygen-Induced Retinopathy Models:

  • Newborn mice are exposed to 75% oxygen from P7 to P12, then returned to normoxia

  • Single 10 μg intravitreal injections of anti-Sema3A antibody or IgG control are administered at P12

  • Parameters measured include:

    • Tip cell density

    • Avascular area measurements

    • Vessel density quantification using retinal flatmounts

• Tumor Angiogenesis Assessment:

  • Anti-Sema3A antibodies (e.g., F11) are administered to GBM patient-derived xenograft models

  • Evaluation includes:

    • Tumor volume measurements

    • Tumor-associated macrophage recruitment analysis

    • Vessel density quantification through immunostaining

    • Expression analysis of angiogenic markers

These analytical approaches provide comprehensive evaluation of how anti-Sema3A antibodies modulate angiogenic processes in various experimental contexts.

What experimental designs are most effective for studying Sema3A's immunomodulatory functions using anti-Sema3A antibodies?

When investigating Sema3A's immunomodulatory functions, the following experimental designs have proven most effective:

• B Cell Regulatory Function Analysis:

  • Flow cytometry to measure Sema3A and NP-1 expression on B cell subsets, particularly CD19+CD25high B regulatory cells

  • Comparison of expression levels between healthy controls and disease groups (e.g., SLE patients)

  • Quantification using both percentage of positive cells and mean fluorescence intensity (MFI)

  B Cell Population	Healthy Controls (Sema3A %)	SLE Patients (Sema3A %)	p-value	MFI (Controls)	MFI (SLE)	p-value
  CD19+CD25high	82.6 ± 6.4%	52.2 ± 5.8%	<0.0001	4.27 ± 0.47	2.68 ± 0.09	0.019
  CD19+CD25low	Lower expression (MFI: 1.8)	Not specified	0.001	Not specified	Not specified	Not specified

• TLR-9 Expression Modulation:

  • Co-culture of CpG-ODN-stimulated B cells from SLE patients with Sema3A

  • Measurement of TLR-9 expression reduction (approximately 50%, P = 0.001)

• Dendritic Cell/T Cell Co-Culture Systems:

  • Addition of anti-Sema3A antibodies to DC/T cell co-cultures

  • Quantification of T cell proliferation responses

  • Comparison with blocking antibodies against endogenous Sema3A

• Cytokine Production Analysis:

  • Assessment of IL-10 expression changes in CD4+NP-1+ T cells following Sema3A exposure

  • Evaluation of regulatory function on effector CD4+ T cells

• In Vivo Disease Models:

  • Administration of anti-Sema3A antibodies in relevant autoimmune disease models

  • Measurement of:

    • Disease activity markers

    • Renal function parameters

    • Autoantibody production

    • Immune cell population distributions

These experimental designs facilitate comprehensive investigation of Sema3A's immunomodulatory roles and the potential therapeutic applications of anti-Sema3A antibodies in autoimmune conditions.

How should researchers validate the specificity of anti-Sema3A antibodies?

Rigorous validation of anti-Sema3A antibody specificity is critical for experimental reliability and reproducibility. Recommended validation protocols include:

• Western Blot Analysis:

  • Test antibody against multiple sample types:

    • Tissue lysates (e.g., neonatal rat brain)

    • Recombinant human Sema3A chimera (95/125 kDa forms)

    • Cell lysates from Sema3A-expressing and non-expressing cells

  • Verify detection of expected molecular weight bands (88.9 kDa for canonical protein, with potential glycosylated forms)

• Cross-Reactivity Testing:

  • Assess binding to related semaphorin family members:

    • Sema3B, Sema3C, Sema3E, Sema3F

    • Use recombinant proteins at equivalent concentrations (0.5-2 μg/ml)

  • Test across species (human, mouse, rat, cynomolgus, rabbit) to determine cross-reactivity profile

• Epitope Mapping:

  • Confirm binding to intended epitope region (e.g., amino acids 103-402 for the A-12 antibody)

  • Utilize peptide competition assays to verify specificity

• Knockout/Knockdown Controls:

  • Apply antibody to samples from Sema3A-knockout models or Sema3A-silenced cells

  • Include isotype controls in all applications (e.g., IgG2b kappa for mouse monoclonals)

• Binding Kinetics Analysis:

  • Surface plasmon resonance to determine KD values

  • Antibody capture assays with injection of various Sema3A proteins (40 μL/min for 600s) and dissociation monitoring (7200s)

• Functional Validation:

  • Confirm neutralizing capability through cell-based assays:

    • Cytoskeletal collapse assays

    • Migration inhibition assays

    • Endothelial permeability assays

Implementing these comprehensive validation steps ensures that experimental outcomes can be confidently attributed to specific Sema3A detection or neutralization.

What are the most effective protocols for using anti-Sema3A antibodies in immunohistochemistry?

Optimized immunohistochemistry protocols for anti-Sema3A antibodies include:

• Tissue Preparation:

  • For paraffin sections: Formalin fixation followed by paraffin embedding

  • Section thickness: 4-5 μm optimal for clear visualization

  • For specialized neural tissue: Consider citric acid treatment for enhanced antigen accessibility

• Antigen Retrieval:

  • Heat-induced epitope retrieval using EDTA buffer pH 9.0 in a pressure chamber (e.g., DAKO PT Link)

  • Alternative: Citrate buffer pH 6.0 for 20 minutes at 95-98°C

  • Cool sections to room temperature before proceeding

• Blocking Steps:

  • Peroxidase block: 3% H₂O₂ in methanol for 10 minutes

  • Protein block: Commercial blocker (e.g., DAKO Protein block) for 10 minutes

  • For high background: Add 5% normal serum from the same species as the secondary antibody

• Primary Antibody Application:

  • Optimal concentration: 1 μg/ml for rabbit polyclonal antibodies

  • Incubation: Overnight at 4°C or 1 hour at room temperature

  • Diluent: PBS with 1% BSA or manufacturer-recommended diluent

• Detection Systems:

  • For chromogenic detection: HRP-conjugated secondary antibody followed by DAB

  • For fluorescent detection: Appropriate fluorophore-conjugated secondary antibody

  • Automated systems (e.g., DAKO Autostainer Plus) can improve reproducibility

• Controls:

  • Positive control: Tissues known to express Sema3A (e.g., cerebellum, dorsal root ganglia)

  • Negative control: Isotype-matched irrelevant antibody at the same concentration

  • Absorption control: Primary antibody pre-incubated with immunizing peptide

• Visualization and Analysis:

  • For brightfield: Counterstain with hematoxylin for nuclear visualization

  • For fluorescence: Include DAPI nuclear counterstain

  • Document staining patterns (cytoplasmic localization is typical for Sema3A)

This optimized protocol enables reliable detection of Sema3A in tissue sections while minimizing background and non-specific staining.

What are the critical factors affecting reproducibility in Sema3A antibody-based assays?

Ensuring reproducibility in Sema3A antibody-based assays requires attention to several critical factors:

• Antibody Selection and Quality:

  • Clone consistency: Use the same clone and lot number when possible

  • Storage conditions: Maintain at recommended temperature (-20°C) and avoid repeated freeze-thaw cycles

  • Aliquoting: Prepare single-use aliquots to preserve antibody integrity

• Sample Preparation Standardization:

  • Consistent lysis buffers for protein extraction

  • Standardized protein quantification methods

  • For serum samples: Use consistent collection, processing, and storage protocols

• Experimental Controls:

  • Include full panel of positive and negative controls

  • For functional assays: Use recombinant Sema3A as a positive control

  • Include isotype controls at equivalent concentrations

• Assay-Specific Considerations:

  • Western blot: Standardize protein loading, transfer conditions, and exposure times

  • ELISA: Validate standard curves with r² > 0.98 and ensure consistent plate washing

  • IHC: Standardize fixation times, antigen retrieval, and development times

• Documentation and Reporting:

  • Record complete antibody information (supplier, catalog number, lot, dilution)

  • Document all experimental conditions in detail

  • Report antibody validation methods used

• Technical Variables:

  • Minimize operator variability through training and standard operating procedures

  • Maintain consistent environmental conditions (temperature, humidity)

  • Calibrate equipment regularly (pipettes, plate readers, imaging systems)

• Statistical Considerations:

  • Perform appropriate power calculations for sample size determination

  • Use multiple technical and biological replicates

  • Apply appropriate statistical tests based on data distribution

Addressing these factors systematically will significantly improve reproducibility of Sema3A antibody-based assays across different laboratories and experimental conditions.

How can researchers optimize anti-Sema3A antibody-based ELISA for serum level quantification?

Optimization of anti-Sema3A antibody-based ELISA for accurate serum quantification involves several methodological refinements:

• Antibody Pair Selection and Validation:

  • Test multiple capture and detection antibody combinations

  • Select pairs recognizing non-overlapping epitopes

  • Validate specificity using recombinant Sema3A and related proteins

  • Recommended dilution for detection antibody: 1:2000

• Protocol Optimization:

  • Coating buffer: Carbonate-bicarbonate buffer (pH 9.6) for optimal protein binding

  • Blocking: 2-3% BSA in PBS to minimize non-specific binding

  • Sample dilution: Test multiple dilutions to ensure measurements within the linear range

  • Incubation times and temperatures: Standardize for consistent results

• Standard Curve Preparation:

  • Use recombinant human Sema3A for standard curve generation

  • Prepare fresh standards for each assay

  • Include 7-8 concentration points with duplicate or triplicate measurements

  • Ensure standard curve range encompasses expected physiological levels (normal range: approximately 74.41 ± 17.60 ng/ml)

• Sample Processing Considerations:

  • Standardize collection procedures (time of day, fasting status)

  • Process serum samples within 2 hours of collection

  • Store aliquots at -80°C to prevent degradation

  • Avoid repeated freeze-thaw cycles

• Quality Control Measures:

  • Include internal controls (low, medium, high) on each plate

  • Calculate intra-assay CV (<10%) and inter-assay CV (<15%)

  • Determine lower limit of detection and quantification

• Data Analysis and Interpretation:

  • Use four-parameter logistic regression for standard curve fitting

  • Apply appropriate statistical analysis for group comparisons

  • Consider potential confounding factors in interpretation:

    • Age and sex matching

    • Medication status

    • Disease duration and activity

This optimized methodology has enabled reliable quantification of serum Sema3A levels in various patient populations, including SLE patients (55.04 ± 16.30 ng/ml) and RA patients (65.54 ± 14.82 ng/ml), allowing for meaningful clinical correlations .

What are the most promising therapeutic applications of anti-Sema3A antibodies based on current research?

Current research highlights several promising therapeutic applications for anti-Sema3A antibodies:

• Glioblastoma (GBM) Treatment:

  • Anti-Sema3A antibodies demonstrate significant tumor inhibitory effects in GBM progression

  • Mechanisms include:

    • Reduction in cell migration and proliferation capabilities of GBM patient-derived cells (PDCs)

    • Down-regulation of cellular proliferative kinetics

    • Inhibition of tumor-associated macrophage recruitment

  • Fully human anti-Sema3A IgG antibodies (F11, A08, C10) show particular promise for clinical translation

• Diabetic Retinopathy:

  • BI-X, a humanized Sema3A antibody, is under investigation for:

    • Proliferative diabetic retinopathy

    • Diabetic macular ischemia

  • Preclinical studies show prevention of:

    • Sema3A-induced endothelial cell permeability

    • Cytoskeletal collapse in human retinal microvascular endothelial cells

  • In oxygen-induced retinopathy models, anti-Sema3A antibodies reduce ischemic avascular area and increase tip cell density

• Autoimmune Disorders:

  • Sema3A's role in immune regulation suggests therapeutic potential in conditions like SLE

  • Exogenous Sema3A can reduce TLR-9 expression on CpG-ODN-stimulated B cells from SLE patients by approximately 50%

  • Anti-Sema3A antibodies may help:

    • Modulate dysregulated Sema3A levels

    • Normalize B cell regulatory functions

    • Potentially attenuate disease activity in specific patient subsets

• Anti-Angiogenic Cancer Therapy:

  • Anti-Sema3A antibodies may counteract tumor resistance to anti-VEGF therapy

  • Sema3A has been implicated in tumor cells' loss of sensitivity to anti-VEGF-targeted therapy

  • Combines with standard chemotherapy for potential synergistic effects

• Neurological Applications:

  • Potential for modulating neuronal growth cone guidance in:

    • Spinal cord injury repair

    • Neuropathic pain management

    • Neurodegenerative conditions

These therapeutic directions are supported by pharmacokinetic and biodistribution studies showing a biological half-life of approximately 6.93 days at 5 mg/kg dosing, making anti-Sema3A antibodies feasible candidates for clinical development .

How can researchers design experiments to investigate the compensatory mechanisms that develop following anti-Sema3A antibody treatment?

Designing experiments to investigate compensatory mechanisms following anti-Sema3A antibody treatment requires sophisticated approaches:

• Temporal Gene Expression Analysis:

  • RNAseq or microarray analysis of treated samples at multiple timepoints (early, mid, and late phases post-treatment)

  • Focus on semaphorin family members and related signaling molecules

  • qRT-PCR validation of differentially expressed genes

  • Compare acute versus chronic anti-Sema3A antibody exposure

• Receptor Expression and Signaling Dynamics:

  • Monitor expression levels of Sema3A receptors (neuropilin-1, neuropilin-2, plexin-A1)

  • Analyze changes in downstream signaling molecules:

    • Small GTPases (Rac1, RhoA)

    • Collapsed response mediator proteins (CRMPs)

    • Cytoskeletal regulators

  • Use phospho-specific antibodies to track activation status of signaling pathways

• Functional Compensation Assessment:

  • Cell Migration Assays:

    • Transwell chamber assays with long-term anti-Sema3A antibody exposure

    • Oris cell migration assay in dose-dependent and time-dependent manner

  • Growth Cone Collapse Studies:

    • Time-course analysis of sensitivity/resistance to Sema3A

    • Cellular impedance measurements following repeated antibody exposure

  • Angiogenesis Models:

    • Repeated administration protocols in oxygen-induced retinopathy

    • Analysis of compensatory angiogenic factor expression

• Combination Treatment Approaches:

  • Test anti-Sema3A antibodies combined with inhibitors of potential compensatory pathways

  • Evaluate synergistic or antagonistic effects through isobologram analysis

  • Analyze tumor response in PDX models under various combination regimens

• Systems Biology Approach:

  • Network analysis of altered signaling pathways

  • Mathematical modeling of compensatory mechanisms

  • Prediction and experimental validation of key compensatory nodes

• In Vivo Longitudinal Studies:

  • Serial sampling in GBM PDX models during extended anti-Sema3A treatment

  • Correlate molecular changes with treatment efficacy over time

  • Identify biomarkers predictive of compensatory resistance

These experimental designs provide comprehensive insights into the adaptive responses to anti-Sema3A therapy, potentially identifying combination strategies to overcome resistance mechanisms.

What methodological approaches can be used to study the interactions between anti-Sema3A antibodies and the neuropilin-1/plexin-1 receptor complex?

Investigating interactions between anti-Sema3A antibodies and the neuropilin-1/plexin-1 receptor complex requires specialized methodological approaches:

• Structural Analysis Techniques:

  • X-ray crystallography of antibody-Sema3A complexes

  • Cryo-electron microscopy to visualize antibody binding to Sema3A in the context of receptor complexes

  • Hydrogen/deuterium exchange mass spectrometry to map binding interfaces

• Binding Kinetics and Competition Assays:

  • Surface plasmon resonance (SPR) to determine:

    • Binding affinities (KD values) between antibodies and Sema3A

    • Competition between antibodies and neuropilin-1/plexin-1 for Sema3A binding

    • Effects of antibodies on Sema3A-receptor complex formation

  • Bio-layer interferometry for real-time, label-free analysis of molecular interactions

• Cellular Interaction Studies:

  • Proximity Ligation Assays (PLA):

    • Visualize and quantify Sema3A-receptor interactions in intact cells

    • Assess how antibodies affect complex formation

  • FRET/BRET Analysis:

    • Fluorescently tag Sema3A and receptors

    • Measure energy transfer in the presence/absence of antibodies

  • Co-immunoprecipitation:

    • Pull down receptor complexes and analyze Sema3A association

    • Determine how antibodies alter complex composition

• Receptor Trafficking and Dynamics:

  • Live-cell imaging with fluorescently tagged components

  • Pulse-chase experiments to track receptor internalization and recycling

  • TIRF microscopy to visualize membrane-proximal events

• Functional Consequence Assessment:

  • Phosphorylation status of downstream signaling molecules

  • Cytoskeletal rearrangements following antibody treatment

  • Growth cone collapse assays in neuronal cultures

  • Endothelial cell permeability assays using FITC-coupled dextran

• Computational Approaches:

  • Molecular dynamics simulations of antibody-Sema3A-receptor interactions

  • In silico epitope mapping and docking studies

  • Prediction of allosteric effects on receptor binding