AAE7 Antibody

What is the EPH Receptor A7 (EPHA7) and why is it important in research?

EPHA7 belongs to the ephrin receptor family, a group of tyrosine kinase receptors involved in cell-cell communication, tissue boundary formation, and neural development. This receptor is particularly significant in research due to its involvement in neurological development, cancer progression, and potential implications in autoimmune conditions. When selecting antibodies against this target, researchers should consider the specific epitope recognition, as different antibodies target different amino acid sequences (such as AA 181-280, 896-925, or 11-41) which may expose different functional domains of interest . The diversity of available binding regions allows for targeted investigation of specific receptor domains and their associated functions in experimental contexts.

How do I select the appropriate EPHA7 antibody for my specific research application?

Selection of the appropriate EPHA7 antibody requires careful consideration of multiple technical parameters. Based on current research methodologies, you should:

• Identify your specific application requirements (Western blotting, immunohistochemistry, flow cytometry, etc.)

• Determine species reactivity needs (human, mouse, rat)

• Consider the epitope region of interest (N-terminal, C-terminal, specific domains)

• Evaluate clonality requirements (monoclonal for high specificity, polyclonal for stronger signals)

• Assess conjugation needs (unconjugated versus fluorophore-conjugated)

For example, if you're investigating EPHA7 in human tissues using immunohistochemistry on paraffin-embedded sections, select an antibody validated for IHC(p) with human reactivity. The search results show several options with different amino acid targeting regions, including polyclonal antibodies targeting regions AA 181-280 with confirmed IHC(p) applications . This antibody would be suitable for visualization of EPHA7 in human tissue sections while avoiding potential cross-reactivity issues with other EPH receptor family members.

What are the key differences between monoclonal and polyclonal EPHA7 antibodies in research applications?

The choice between monoclonal and polyclonal EPHA7 antibodies significantly impacts experimental outcomes:

What are the validated applications for commonly used EPHA7 antibodies?

Current research demonstrates EPHA7 antibodies have been validated across multiple experimental platforms with application-specific considerations:

How should I optimize Western blotting protocols when using EPHA7 antibodies?

Optimization of Western blotting protocols for EPHA7 detection requires several methodological considerations:

• Sample preparation:

  • Use RIPA buffer supplemented with phosphatase inhibitors (especially important when studying phosphorylated forms like pTyr791)

  • Include protease inhibitor cocktail to prevent receptor degradation

  • Denature at 95°C for 5 minutes in reducing sample buffer

• Gel selection and transfer:

  • Use 7-8% gels for optimal resolution of the full-length receptor (~130 kDa)

  • Transfer proteins to PVDF membranes (preferred over nitrocellulose for this high molecular weight protein)

  • Extend transfer time to 2 hours or use overnight cold transfer for complete migration

• Antibody incubation:

  • For polyclonal antibodies (like AA 181-280), use 1:500-1:1000 dilution

  • Extend primary antibody incubation to overnight at 4°C

  • Include 5% BSA in TBST for blocking and antibody dilution to reduce background

• Signal detection optimization:

  • Use enhanced chemiluminescence with extended exposure times for weak signals

  • Consider signal amplification systems for low abundance detection

These optimizations are particularly important when working with clinical samples or low-expressing cell lines, where sensitivity is critical. Researchers should validate antibody specificity using positive control lysates from cells known to express EPHA7, and consider including knockout/knockdown controls to confirm band specificity .

How can I address potential cross-reactivity issues with EPHA7 antibodies?

Cross-reactivity is a significant concern with EPHA7 antibodies due to the high sequence homology within the EPH receptor family. The research data indicates a 73% non-sequential sequence similarity between EPHA7 and EPHA4, creating potential specificity challenges . To address these issues:

• Validation strategies:

  • Perform parallel experiments with EPHA4-specific antibodies to identify potential cross-reactivity

  • Include negative controls (tissues/cells known to lack EPHA7 expression)

  • Use genetic approaches (siRNA knockdown of EPHA7) to confirm antibody specificity

  • Compare staining patterns with multiple antibodies targeting different EPHA7 epitopes

• Technical approaches to minimize cross-reactivity:

  • Increase antibody dilution to reduce non-specific binding

  • Extend washing steps in immunoassays

  • Pre-absorb antibodies with recombinant EPHA4 protein when cross-reactivity is a concern

  • For immunohistochemistry, implement antigen retrieval optimization

• Analytical considerations:

  • Always include appropriate isotype controls

  • When possible, confirm antibody findings with complementary techniques (e.g., mRNA analysis)

  • Consider using subtype-specific antibodies that target unique regions with minimal homology

This methodological approach parallels best practices seen in other receptor antibody validation studies, where careful consideration of cross-reactivity is essential for generating reliable research data .

What are the predicted species cross-reactivities for commonly used EPHA7 antibodies?

Current research data indicates varying species cross-reactivity profiles for EPHA7 antibodies:

For researchers conducting comparative or translational studies across species, the AA 181-280 antibody offers the most versatile application profile with confirmed reactivity in common laboratory species (human, mouse, rat) and predicted reactivity in additional mammals . When working with non-standard research animals, researchers should perform preliminary validation studies to confirm cross-reactivity before proceeding with full experimental designs.

How can EPHA7 antibodies be utilized in high-avidity, low-affinity (HALA) approaches for targeted therapeutic development?

EPHA7 antibodies can be engineered for High Avidity, Low Affinity (HALA) applications, representing an advanced research frontier for targeted therapeutic development. This approach offers significant advantages for precision medicine applications, particularly for targets with variable expression levels across tissues:

• Mechanism of action:

  • HALA antibodies utilize weaker monovalent binding affinity but stronger avidity (multi-point binding)

  • This creates a concentration-dependent competitive binding scenario where HALA antibodies preferentially compete with therapeutic antibody-drug conjugates (ADCs) in high-expression tissues

  • In low-expression tissues, the ADC outcompetes the HALA antibody, allowing targeted binding

• Implementation methodology:

  • Engineer EPHA7 antibodies with specific affinity modifications (typically 10-100 fold lower KD than the therapeutic antibody)

  • Maintain bivalent binding capacity to preserve avidity effects

  • Administer as a pre-treatment before therapeutic antibody administration

• Optimization parameters:

  • Thiele modulus (a dimensionless number describing diffusion-reaction relationships)

  • Competition number (ratio of HALA and therapeutic antibody competitive binding)

  • Concentration optimization through computational modeling

This approach could be particularly valuable for EPHA7-targeted therapies in cancer research, where expression levels vary significantly between tumor types and between patients. The HALA methodology provides an in situ adjustment mechanism that potentially improves tissue penetration and efficacy of EPHA7-targeted therapeutics .

What role do EPHA7 antibodies play in autoimmune encephalitis and neural antibody testing?

Emerging research suggests potential applications for EPHA7 antibodies in the context of autoimmune neurological conditions:

• Diagnostic applications:

  • Neural-specific antibodies serve as critical biomarkers in autoimmune encephalitis

  • The incidence of antibody-positive autoimmune encephalitis has nearly tripled from 2006-2015 compared to 1995-2005

  • Worldwide, approximately one million patients have autoimmune encephalitis, with 90,000 new cases annually

• Clinical and paraclinical features:

  • T2 FLAIR hyperintensity in medial temporal lobes is associated with certain neural antibodies

  • Specific antibodies correlate with differential treatment responses and prognosis

  • High APE2 scores (Antibody Prevalence in Epilepsy) correlate with detection of neural-specific antibodies of clinical relevance

• Methodological considerations:

  • Cerebrospinal fluid and serum testing provide complementary information

  • Immunohistochemistry patterns may help identify novel antigenic targets

  • Multiple antibody testing is often required due to overlapping clinical presentations

While the specific role of EPHA7 in autoimmune encephalitis requires further investigation, the methodological approaches for neural antibody testing provide a framework for studying potential EPHA7 involvement in neurological autoimmunity .

What are common technical challenges when using EPHA7 antibodies and how can they be addressed?

Researchers frequently encounter technical challenges when working with EPHA7 antibodies across various experimental platforms:

A methodical approach to troubleshooting involves systematically varying one parameter at a time while maintaining others constant. For particularly challenging applications, consider comparing multiple antibodies targeting different EPHA7 epitopes to validate findings .

How should I validate EPHA7 antibody specificity for critical research applications?

Validation of EPHA7 antibody specificity is essential for generating reliable research data, particularly in publication-quality experiments:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout of EPHA7 in relevant cell lines

  • siRNA knockdown with quantitative assessment of protein reduction

  • Overexpression systems with tagged EPHA7 variants

• Biochemical validation:

  • Peptide competition assays using the immunizing peptide

  • Immunoprecipitation followed by mass spectrometry

  • Parallel testing with multiple antibodies targeting different epitopes

  • Western blot analysis to confirm expected molecular weight

• Application-specific validation:

  • For immunohistochemistry: Compare staining patterns with in situ hybridization data

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

  • For ELISA: Establish standard curves with recombinant protein

  • For immunofluorescence: Include appropriate subcellular markers to confirm expected localization

• Disease-relevant validation:

  • Compare antibody performance in normal versus disease tissues

  • Correlate protein detection with known transcriptional changes in disease models

  • Verify detection in the species and tissue of interest

This comprehensive validation approach aligns with current best practices in antibody-based research and is especially important when investigating targets like EPHA7 that share significant homology with related family members .

How are EPHA7 antibodies being utilized in antibody-drug conjugate (ADC) development?

EPHA7 antibodies are finding novel applications in antibody-drug conjugate (ADC) development, particularly leveraging the receptor's differential expression patterns across tissues:

• ADC design considerations:

  • Selection of antibody binding domains with optimal tumor penetration

  • Conjugation chemistry optimization for payload delivery

  • Linker selection based on cellular internalization kinetics

  • Payload mechanism selection (e.g., topoisomerase inhibitors versus other cytotoxic agents)

• Targeting strategy optimization:

  • Utilization of computational models to predict tissue penetration

  • Application of bivalent competition kinetic models for competitive binding assessment

  • Integration of target internalization dynamics into efficacy models

  • Consideration of bystander effects for hydrophilic versus lipophilic payloads

• Clinical translation considerations:

  • Expression profiling across tumor types to identify appropriate indications

  • Assessment of normal tissue expression for toxicity prediction

  • Development of companion diagnostics for patient selection

The HALA antibody approach described earlier represents a particularly promising direction for EPHA7-targeted ADCs, potentially allowing for improved tumor penetration while minimizing off-target effects in tissues with high EPHA7 expression .

What are the key considerations for interpreting contradictory results from different EPHA7 antibodies?

When faced with contradictory results using different EPHA7 antibodies, researchers should implement a systematic analysis framework:

• Epitope-based analysis:

  • Map the specific binding regions of each antibody (e.g., AA 181-280 vs. AA 896-925)

  • Consider potential differential detection of EPHA7 isoforms or processed forms

  • Evaluate epitope accessibility in different experimental conditions

  • Assess whether phosphorylation status affects antibody recognition

• Technical comparison:

  • Evaluate differences in antibody format (monoclonal vs. polyclonal)

  • Compare antibody production methods and immunization strategies

  • Consider differences in antibody concentration, incubation time, and detection methods

  • Analyze potential differences in sample preparation affecting epitope exposure

• Validation framework:

  • Implement orthogonal techniques to confirm biological findings

  • Utilize genetic approaches (knockdown/knockout) to resolve antibody specificity

  • Consider potential cross-reactivity with homologous proteins (e.g., EPHA4)

  • Determine expression levels that may influence detection sensitivity

• Biological context interpretation:

  • Consider cell/tissue-specific post-translational modifications

  • Evaluate protein-protein interactions that may mask specific epitopes

  • Analyze subcellular localization differences that may affect antibody accessibility

By systematically analyzing these factors, researchers can reconcile apparently contradictory results and gain deeper insights into the biological complexity of EPHA7 expression and function .