EPHA10 Antibody, HRP conjugated

What is EPHA10 and why is it a valuable target for cancer research?

EPHA10 (ephrin receptor A10) is a receptor tyrosine kinase that has gained significant attention as a cancer biomarker due to its unique expression pattern. It is virtually undetectable in most normal tissues except the male testis, yet shows high expression in several malignancies . This selective expression profile makes it an ideal target for cancer therapeutics with potentially minimal adverse effects on normal tissues.

Research has demonstrated that EPHA10 expression correlates with tumor progression and poor prognosis in several cancer types, including triple-negative breast cancer (TNBC) . It has been significantly linked to lymph node metastasis and higher tumor staging in human breast cancer specimens . Additionally, EPHA10 is expressed not only in cancer cells but also in immunosuppressive myeloid cells within the tumor microenvironment, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) .

What are the key applications for EPHA10 antibodies in research settings?

EPHA10 antibodies serve multiple critical research applications:

• Detection and Localization: For identifying EPHA10 expression in tissue samples and cell lines using techniques such as immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry .

• Protein Analysis: For quantifying and characterizing EPHA10 protein via Western blotting and ELISA .

• Therapeutic Development: As potential direct therapeutic agents or as components of targeted delivery systems for cancer treatment .

• CAR-T Cell Therapy: For developing chimeric antigen receptor T cells that specifically target EPHA10-expressing tumors .

HRP-conjugated EPHA10 antibodies particularly enhance detection sensitivity in applications such as Western blotting, ELISA, and IHC through enzymatic signal amplification.

How does EPHA10 expression differ between normal and cancerous tissues?

EPHA10 exhibits a highly restricted expression pattern that makes it particularly valuable as a cancer target. In normal tissues, EPHA10 expression is essentially undetectable except in male testis tissue . This limited normal tissue distribution suggests that EPHA10-targeted therapies may have minimal off-target effects.

In contrast, EPHA10 shows significantly elevated expression in the tumor regions of multiple cancer types. Research using human tissue microarrays has demonstrated that EPHA10 expression levels are significantly higher in breast, lung, and ovarian cancer tumor regions compared to adjacent normal tissues . Within the tumor microenvironment, EPHA10 is expressed both by cancer cells and by immunosuppressive myeloid cells including TAMs and MDSCs, but not by tumor-infiltrating T cells as confirmed by immunofluorescence staining .

What is the optimal protocol for using HRP-conjugated EPHA10 antibodies in immunohistochemistry?

For optimal IHC results with HRP-conjugated EPHA10 antibodies, follow this methodological approach:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section at 4-5 μm thickness onto positively charged slides

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Allow slides to cool to room temperature for 20 minutes

• Blocking and Primary Antibody Incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal serum for 30 minutes

  • Apply HRP-conjugated EPHA10 antibody at optimized dilution (typically 1:100 to 1:500) and incubate at 4°C overnight

• Detection and Visualization:

  • Since the antibody is HRP-conjugated, no secondary antibody is required

  • Develop signal with DAB substrate for 5-10 minutes (optimize timing based on signal intensity)

  • Counterstain with hematoxylin, dehydrate, and mount

• Controls:

  • Include EPHA10-positive tissues (breast cancer or testis) as positive controls

  • Include normal tissues (except testis) as negative controls

  • Consider including absorption controls to confirm specificity

For multiplexed staining, carefully consider that EPHA10 co-localizes with TAMs and MDSCs but not with T cells in the tumor microenvironment .

How can I validate the specificity of my HRP-conjugated EPHA10 antibody?

Validating antibody specificity is crucial for generating reliable research data. For HRP-conjugated EPHA10 antibodies, implement these complementary validation approaches:

• Positive and Negative Tissue Controls:

  • Test on tissues known to express EPHA10 (breast, lung, or ovarian cancer samples; testis)

  • Confirm absence of staining in tissues known to lack EPHA10 expression (most normal tissues)

• Competitive Binding Assay:

  • Pre-incubate the antibody with recombinant EPHA10 protein before application

  • Specific antibodies will show reduced or eliminated staining

• Cellular Models:

  • Test on EPHA10-overexpressing cell lines versus controls

  • Compare with EPHA10 knockout or knockdown models

  • Research has shown specific binding to EPHA10-expressing cells like BT-549 with no binding to mock controls

• Cross-Reactivity Assessment:

  • Test for cross-reactivity with other EphA family members (EphA1-EphA8)

  • Well-validated EPHA10 antibodies have demonstrated specific binding to EPHA10 without cross-reacting with other EphA isoforms

• Multiple Detection Methods:

  • Correlate IHC results with other detection methods such as Western blot, RT-PCR, or flow cytometry

For rigorous validation, a flow cytometry-based assay comparing fluorescence intensity between cells expressing human EPHA10 and mock controls can effectively demonstrate antibody specificity, as shown with other EPHA10 antibodies .

What are the optimal conditions for Western blotting using HRP-conjugated EPHA10 antibodies?

For optimal Western blotting results with HRP-conjugated EPHA10 antibodies:

• Sample Preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration using BCA or Bradford assay

  • Load 20-50 μg of protein per lane

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE (EPHA10 is ~120 kDa)

  • Include positive control lysates from EPHA10-expressing cancer cells

• Transfer:

  • Transfer to PVDF membrane (recommended over nitrocellulose for EPHA10)

  • Use wet transfer at 100V for 90 minutes or 30V overnight at 4°C

• Blocking and Antibody Incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute HRP-conjugated EPHA10 antibody in blocking buffer (typically 1:1000 to 1:5000)

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • Since the antibody is HRP-conjugated, proceed directly to detection

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or use digital imaging system

• Optimization Notes:

  • EPHA10 may require extended transfer times due to its high molecular weight

  • If background is high, increase washing time or add 0.1% Tween-20 to blocking buffer

  • For validation, compare results across multiple EPHA10-expressing and non-expressing samples

How can HRP-conjugated EPHA10 antibodies be used to study the tumor microenvironment?

HRP-conjugated EPHA10 antibodies offer powerful tools for investigating the tumor microenvironment (TME) when used in these methodological contexts:

• Multiplex Immunohistochemistry:

  • Combine with antibodies against immune cell markers to identify EPHA10 expression patterns

  • Sequential staining protocols using HRP-conjugated EPHA10 antibody followed by tyramide signal amplification and antibody stripping

  • This approach can reveal co-localization of EPHA10 with immunosuppressive myeloid cells such as TAMs (F4/80+/CD163+) and MDSCs (CD11b+/Gr-1+)

• Tissue Microarray Analysis:

  • Apply to cancer tissue microarrays to assess correlation between EPHA10 expression and:

    • Infiltration of different immune cell populations

    • Expression of immune checkpoint molecules (e.g., PD-L1)

    • Patient outcomes and treatment responses

• Dual Chromogenic IHC:

  • Use HRP-conjugated EPHA10 antibodies with other enzyme-conjugated antibodies (e.g., alkaline phosphatase)

  • Visualize with contrasting chromogens (DAB for EPHA10, Fast Red for other markers)

  • Quantify co-expression using digital image analysis

Research has shown that EPHA10 expression correlates with increased PD-L1 expression and immunosuppression , suggesting its role in immune evasion mechanisms. When analyzing the TME, focus particularly on the co-localization of EPHA10 with TAMs and MDSCs, which are known to suppress antitumor immunity through various mechanisms .

What strategies can optimize EPHA10 antibody-based targeted therapeutic delivery systems?

Developing effective EPHA10 antibody-based targeted delivery systems requires careful optimization of several parameters:

• Antibody Conjugation Chemistry:

  • For liposomal delivery systems, conjugate anti-EPHA10 antibodies to liposomes using EDCI and sulfo-NHS chemistry in pH 7.4 PBS with 2-hour incubation at room temperature

  • For nanoparticle systems, consider maleimide-thiol coupling after antibody reduction

  • Purify conjugated systems using gel filtration on Sepharose 4B columns equilibrated in PBS

• Target Binding and Specificity:

  • Select antibody clones with high specificity for EPHA10 that do not cross-react with other EphA family members

  • Prioritize clones that demonstrate strong cell surface binding in flow cytometry assays

  • Consider antibodies that recognize epitopes in the extracellular domain of EPHA10

• Payload Considerations:

  • For siRNA delivery, develop pH-sensitive liposomes modified with cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)

  • For toxic payloads, calculate optimal drug-to-antibody ratios to maintain binding while maximizing efficacy

  • Consider stimuli-responsive release mechanisms (pH, redox, enzymatic) based on tumor microenvironment

• In Vivo Validation:

  • Perform antibody chasing assays with fluorescently labeled antibodies in xenograft models to confirm tumor-specific accumulation

  • Monitor pharmacokinetics and biodistribution to ensure minimal accumulation in non-target organs

  • Assess therapeutic efficacy in appropriate syngeneic models that maintain intact immune systems

Research has demonstrated that anti-EPHA10 antibodies can specifically accumulate in tumor regions according to EPHA10 levels without significant accumulation in other organs, making them excellent candidates for targeted delivery systems .

How does EPHA10 antibody targeting affect T cell-mediated antitumor immunity?

EPHA10 antibody targeting significantly impacts T cell-mediated antitumor immunity through multiple mechanisms:

• Direct Effects on Immunosuppressive Myeloid Cells:

  • EPHA10 is expressed on immunosuppressive myeloid cells including TAMs and MDSCs, but not on T cells

  • Anti-EPHA10 antibody treatment likely modulates these immunosuppressive cells, potentially reducing their suppressive functions

  • This indirect mechanism helps restore T cell function in the tumor microenvironment

• Enhancement of CTL-Mediated Responses:

  • Studies with anti-EPHA10 mAb clone #4 have demonstrated enhanced tumor regression in syngeneic TNBC mouse models

  • This was accompanied by improved CTL-mediated antitumor immunity without significant toxicity

  • The therapeutic effect appears to be dose-dependent, with higher doses (300 μg/mouse vs. 150 μg/mouse) showing greater efficacy

• Relationship with Immune Checkpoint Pathways:

  • Higher EPHA10 expression has been associated with increased PD-L1 expression and immunosuppression

  • Targeting EPHA10 may downregulate PD-L1 expression, potentially complementing immune checkpoint inhibitor therapies

• CAR-T Cell Approaches:

  • EPHA10-specific CAR-T cells derived from anti-EPHA10 antibodies (e.g., clone #4) significantly inhibit TNBC cell viability in vitro and tumor growth in vivo

  • This approach directly harnesses T cell cytotoxicity against EPHA10-expressing tumors

The efficacy of anti-EPHA10 therapy in improving antitumor immunity has been demonstrated across multiple syngeneic mouse models of TNBC, including 4T1 and EMT6, suggesting broad applicability across different TNBC subtypes .

What are common issues when using HRP-conjugated EPHA10 antibodies and how can they be resolved?

When working with HRP-conjugated EPHA10 antibodies, researchers may encounter several technical challenges. Here are methodological solutions to common problems:

For particularly challenging samples, consider using amplification systems such as biotin-streptavidin or tyramide signal amplification to enhance detection sensitivity without increasing background.

How can I determine the optimal concentration of HRP-conjugated EPHA10 antibody for different applications?

Determining optimal antibody concentration requires systematic titration across different applications. Follow these methodological approaches:

• For Western Blotting:

  • Prepare a titration series ranging from 1:500 to 1:5000 dilution

  • Use positive control lysates (EPHA10-expressing cancer cells) alongside negative controls

  • Select the dilution that provides clear specific bands with minimal background

  • For novel samples, consider starting with a broader range (1:100 to 1:10,000)

• For Immunohistochemistry/Immunofluorescence:

  • Prepare a dilution series from 1:50 to 1:500

  • Apply to serial sections of known positive tissue (breast cancer or testis)

  • Evaluate for:

    • Signal-to-noise ratio

    • Specificity of staining pattern (membrane localization)

    • Correlation with expected expression patterns

  • The optimal dilution shows specific staining with minimal background

• For ELISA:

  • Perform a checkerboard titration using:

    • Coating antigen at 0.1-10 μg/mL

    • Antibody dilutions from 1:100 to 1:10,000

  • Generate standard curves at each antibody concentration

  • Select the dilution providing the widest dynamic range with lowest EC50

• For Flow Cytometry:

  • Test dilutions from 1:50 to 1:500 on positive cells (e.g., BT-549 expressing human EPHA10) versus negative controls

  • Measure median fluorescence intensity and calculate signal-to-noise ratio

  • Select the dilution with highest specific signal and minimal background

Each new lot of antibody should undergo re-titration, as conjugation efficiency may vary between manufacturing batches. Document optimal conditions for reproducibility across experiments.

What are the best practices for storage and handling of HRP-conjugated EPHA10 antibodies to maintain activity?

Proper storage and handling of HRP-conjugated antibodies is critical for maintaining enzymatic activity and binding specificity. Follow these evidence-based practices:

• Storage Conditions:

  • Store antibody at -20°C for long-term storage (up to 1 year)

  • For working stocks, store at 4°C for up to 1 month

  • Add 50% glycerol to stock solutions to prevent freeze-thaw damage

  • Include preservatives (0.02% sodium azide or 0.1% thimerosal) for stocks stored at 4°C

  • Note: Sodium azide inhibits HRP activity, so dilute sufficiently before use

• Aliquoting Strategy:

  • Prepare single-use aliquots (10-50 μL) upon receipt

  • Use siliconized or low-protein binding tubes

  • Label with date, concentration, and freeze-thaw count

  • Never refreeze thawed aliquots

• Thawing Protocol:

  • Thaw rapidly at room temperature with gentle agitation

  • Do not use heat sources as they may denature the antibody

  • Centrifuge briefly after thawing to collect solution

  • Use immediately after thawing for optimal results

• Dilution Guidelines:

  • Use freshly prepared buffers for dilutions

  • Include carrier proteins (0.1-1% BSA) in dilution buffers

  • For most applications, prepare dilutions immediately before use

  • For multi-day experiments, prepare fresh dilutions daily

• Stability Monitoring:

  • Include consistent positive controls in each experiment

  • Monitor signal intensity over time as indicator of stability

  • Consider including HRP activity controls (commercial standards)

  • If signal decreases >30% from baseline, use a new aliquot

• Environmental Factors:

  • Protect from extended exposure to light

  • Avoid repeated temperature fluctuations

  • Maintain pH between 6.0-8.0 for optimal HRP stability

  • Avoid oxidizing agents and metal ions

Following these practices will help maintain antibody performance, ensure experimental reproducibility, and maximize the usable lifetime of valuable HRP-conjugated EPHA10 antibodies.

How might EPHA10 antibodies be integrated into combination immunotherapy approaches?

EPHA10 antibodies hold significant potential for integration into combination immunotherapy strategies through several mechanistic pathways:

• Combination with Immune Checkpoint Inhibitors:

  • EPHA10 expression correlates with increased PD-L1 expression and immunosuppression

  • Anti-EPHA10 therapy could synergize with PD-1/PD-L1 blockade by:

    • Enhancing T cell infiltration into tumor sites

    • Reducing immunosuppressive myeloid cells (TAMs and MDSCs) that express EPHA10

    • Potentially downregulating PD-L1 expression on tumor cells

  • Design sequential treatment protocols to prime the immune environment with anti-EPHA10 before checkpoint inhibition

• Enhancement of CAR-T Cell Approaches:

  • Develop dual-targeting strategies combining:

    • EPHA10-specific CAR-T cells for direct tumor targeting

    • Anti-EPHA10 antibodies to modulate the immunosuppressive microenvironment

  • This approach could potentially overcome resistance mechanisms by addressing both tumor cells and their protective microenvironment simultaneously

• Antibody-Drug Conjugates (ADCs) with Immunomodulatory Payloads:

  • Engineer anti-EPHA10 ADCs carrying payloads that can:

    • Directly kill EPHA10+ tumor cells

    • Release immunostimulatory molecules into the tumor microenvironment

    • Deplete immunosuppressive cell populations

  • These "immunomodulatory ADCs" could serve as bridges between targeted therapy and immunotherapy

• Combination with Cancer Vaccines:

  • Anti-EPHA10 antibody treatment enhances CTL-mediated antitumor immunity

  • This effect could amplify responses to cancer vaccines by:

    • Reducing immunosuppression in the tumor microenvironment

    • Potentially releasing tumor antigens through antibody-dependent cellular cytotoxicity

    • Creating a more favorable environment for vaccine-induced T cells

Research in syngeneic TNBC mouse models has already demonstrated that anti-EPHA10 antibody treatment enhances tumor regression and improves therapeutic response rates , providing a strong rationale for combination approaches.

What methodological approaches can help identify the mechanisms of EPHA10-mediated immunosuppression?

Elucidating the mechanisms of EPHA10-mediated immunosuppression requires sophisticated experimental approaches:

• Single-Cell RNA Sequencing of the Tumor Microenvironment:

  • Isolate cells from EPHA10-high versus EPHA10-low tumors

  • Perform scRNA-seq to identify:

    • Transcriptional differences in immune cell populations

    • Altered signaling pathways in EPHA10-expressing cells

    • Changes in immunosuppressive molecule expression

  • Correlate findings with spatial information using spatial transcriptomics

• Functional Assays of Myeloid-T Cell Interactions:

  • Isolate EPHA10+ myeloid cells (TAMs and MDSCs) from tumors

  • Co-culture with T cells to assess:

    • T cell proliferation (CFSE dilution assay)

    • Cytokine production (intracellular cytokine staining)

    • Cytotoxic activity against tumor targets

  • Compare effects of anti-EPHA10 antibody treatment on these interactions

• Phospho-Proteomic Analysis:

  • Perform phospho-proteomics on EPHA10+ cells with and without antibody treatment

  • Map EPHA10 signaling networks and their connection to immunosuppressive pathways

  • Identify key nodes that could be targeted simultaneously with EPHA10

• Genetic Manipulation Approaches:

  • Generate EPHA10 knockout or knockdown in specific cell populations using:

    • Conditional knockout mouse models

    • Cell type-specific CRISPR-Cas9 delivery

  • Compare with effects of antibody blockade to identify:

    • On-target versus off-target effects

    • Signaling-dependent versus independent mechanisms

• In Vivo Imaging of Immune Cell Dynamics:

  • Utilize intravital microscopy to visualize:

    • T cell trafficking in EPHA10-high versus EPHA10-low tumors

    • Interactions between EPHA10+ myeloid cells and T cells

    • Effects of anti-EPHA10 antibody treatment on these dynamics

The finding that EPHA10 co-localizes with immunosuppressive myeloid cells (TAMs and MDSCs) but not with T cells in the tumor microenvironment provides a critical starting point for these mechanistic investigations.

What innovations might improve the specificity and efficacy of EPHA10-targeted therapeutic approaches?

Several innovative approaches could enhance the specificity and efficacy of EPHA10-targeted therapeutics:

• Bispecific Antibody Platforms:

  • Develop bispecific antibodies targeting:

    • EPHA10 on tumor cells and CD3 on T cells to redirect T cell cytotoxicity

    • EPHA10 and immunosuppressive receptors (e.g., PD-L1) for dual checkpoint blockade

    • EPHA10 and myeloid cell markers to specifically target immunosuppressive populations

  • Optimize affinity and geometry for maximal efficacy with minimal adverse effects

• Advanced Antibody Engineering:

  • Generate site-specific conjugation methods for ADCs to maintain binding properties

  • Develop antibody fragments (Fabs, scFvs) for improved tumor penetration

  • Engineer Fc modifications to enhance:

    • Antibody-dependent cellular cytotoxicity (ADCC)

    • Complement-dependent cytotoxicity (CDC)

    • Extended half-life through FcRn interactions

• Responsive Nanoparticle Delivery Systems:

  • Create pH-sensitive liposomes conjugated with anti-EPHA10 antibodies that:

    • Remain stable in circulation

    • Release payloads specifically in the acidic tumor microenvironment

    • Can deliver combination therapeutics (e.g., siRNA plus small molecules)

  • Incorporate stimuli-responsive elements that respond to tumor-specific triggers

• Improved CAR-T Cell Designs:

  • Develop next-generation EPHA10-specific CAR-T cells with:

    • Inducible expression systems for safety

    • Additional costimulatory domains for enhanced persistence

    • Resistance to exhaustion in the immunosuppressive TME

    • Logic-gated activation requiring multiple tumor antigens

• Combination with Epigenetic Modifiers:

  • Investigate whether epigenetic drugs can increase EPHA10 expression in:

    • Cancer stem cells that may have lower expression

    • Tumor cells that have lost expression as an escape mechanism

  • This approach could expand the population of targetable cells

Research has already demonstrated that anti-EPHA10 mAbs with high specificity for EPHA10 (without cross-reactivity to other EphA family members) can effectively target tumor regions in vivo . Building on this foundation with these innovative approaches could significantly enhance therapeutic outcomes.