ERF010 Antibody

What is the target specificity of ERF010 antibody and how is it validated?

ERF010 antibody targets specific epitopes on receptor tyrosine kinases, similar to anti-EphA10 monoclonal antibodies that recognize cell surface receptors without cross-reactivity with other family isoforms. Validation typically employs multiple techniques:

• Flow cytometry to assess binding to cell-surface receptors

• Enzyme-linked immunosorbent assay (ELISA) to evaluate binding affinity and specificity

• Immunofluorescence to confirm targeting of tumor regions

• Western blotting to verify target protein expression

Proper validation includes comparison with isotype controls and testing against multiple cell lines with varying target expression levels. Researchers should verify that the antibody recognizes the intended target without cross-reactivity to closely related proteins, especially important for receptor families with high sequence homology.

What are the recommended applications for ERF010 antibody in cancer research?

ERF010 antibody can be utilized in multiple cancer research applications, similar to other receptor tyrosine kinase antibodies:

• Immunohistochemistry to analyze expression patterns in tumor tissues

• Flow cytometry for quantifying receptor expression on cancer cells

• In vivo tumor targeting studies

• Development of chimeric antigen receptor (CAR) T cell therapies

• Studies of tumor microenvironment interactions

For optimal results in immunohistochemistry of paraffin-embedded tissues, dilutions between 1/250 and 1/500 are typically effective, though optimization may be required for specific tissue types .

What expression patterns should researchers expect when using ERF010 antibody?

Based on studies with similar receptor tyrosine kinase antibodies, researchers should expect:

• High expression in tumor regions of certain cancer types (breast, lung, ovarian cancers)

• Expression in immunosuppressive myeloid cells within the tumor microenvironment

• Limited expression in normal adult tissues, with possible exception of testicular tissue

• Co-localization with tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs)

When conducting immunofluorescence studies, researchers may observe co-localization patterns with F4/80, CD163, CD11b, and Gr-1 markers, which would indicate expression in myeloid compartments of the tumor microenvironment.

How can ERF010 antibody be used in developing targeted immunotherapies?

ERF010 antibody can be leveraged for immunotherapy development through multiple approaches:

• Antibody-based therapeutics: The antibody can be used directly as a therapeutic agent, particularly if it demonstrates tumor regression capabilities in preclinical models. In studies with EphA10 monoclonal antibodies, treatment resulted in 40% response rates under therapeutic categories of stable disease and partial/complete response in triple-negative breast cancer (TNBC) models .

• CAR-T cell development: The antibody's binding domain can be incorporated into chimeric antigen receptor constructs for T cell engineering. This approach has shown promising results in inhibiting tumor cell viability in vitro and tumor growth in vivo for similar receptor-targeting antibodies .

• Combination therapies: Researchers can explore synergistic effects by combining ERF010 antibody with checkpoint inhibitors or other immunomodulatory agents, particularly since receptor tyrosine kinases may have immunosuppressive effects in the tumor microenvironment.

What are the optimal imaging parameters for structural characterization of ERF010 antibody-antigen complexes?

For structural characterization using cryoEM, researchers should consider the following parameters:

Researchers should aim for near-atomic resolution (~3-4 Å) to accurately characterize antibody-antigen interactions. Software packages like UCSF Chimera can be used for visualization of EM density maps .

How can researchers address epitope-specific binding inconsistencies in experimental results with ERF010 antibody?

When encountering binding inconsistencies:

• Perform epitope mapping: Use techniques such as HDX-MS (hydrogen-deuterium exchange mass spectrometry) or alanine scanning mutagenesis to precisely identify the binding epitope.

• Evaluate conformational dependencies: Some antibodies recognize conformational epitopes that may be sensitive to experimental conditions. Test binding under various pH, salt concentrations, and temperatures.

• Assess glycosylation impact: If the target is glycosylated, test whether enzymatic deglycosylation affects antibody binding.

• Consider allosteric effects: Some antibodies may exhibit different binding properties depending on whether the receptor is in an active or inactive conformation, or if it has bound its natural ligand.

• Validate with orthogonal methods: Complement binding studies with surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to get quantitative binding parameters under controlled conditions .

What are the recommended protocols for validating ERF010 antibody specificity across multiple experimental platforms?

A comprehensive validation approach should include:

Flow Cytometry Validation:

• Use cell lines with confirmed high and low/no expression of the target

• Include appropriate isotype controls

• Test at multiple antibody concentrations (typically 1-10 μg/ml)

• Compare fluorescence intensity between target-positive and negative cells

ELISA Validation:

• Coat plates with the purified target protein and closely related family members

• Test antibody binding to all proteins under identical conditions

• Develop a standard curve using known concentrations

• Calculate cross-reactivity percentages

Immunohistochemistry Validation:

• Use positive control tissues with known expression

• Include negative control tissues

• Test multiple antibody dilutions (starting at 1/500)

• Compare with alternative antibodies targeting the same protein

Western Blot Validation:

• Run samples from multiple tissue/cell types

• Include recombinant protein standards

• Verify band size matches predicted molecular weight

• Perform peptide competition assays to confirm specificity

What are the critical parameters for developing ERF010 antibody-derived CAR-T cells?

When developing CAR-T cells using ERF010 antibody-derived binding domains:

Antibody Fragment Selection:

• Single-chain variable fragments (scFv) derived from the antibody should maintain the specificity and affinity of the parent antibody

• Test multiple orientations (VH-VL vs. VL-VH) as this can affect CAR expression and function

• Consider using alternative binding domains such as nanobodies if size is a concern

CAR Design Considerations:

• Optimal spacer length must be determined empirically, as it affects the immunological synapse formation

• Evaluate multiple co-stimulatory domains (CD28, 4-1BB, OX40) for persistence and efficacy

• Consider incorporating safety switches (e.g., suicide genes) for clinical applications

Functional Testing:

• Measure cytokine production (IFN-γ, TNF-α, IL-2) upon target recognition

• Evaluate cytotoxicity against target-positive and negative cell lines

• Assess persistence in mouse models

• Monitor for off-target effects in in vivo models

What troubleshooting strategies are recommended for inconsistent ERF010 antibody performance in immunohistochemistry?

When encountering inconsistent results in immunohistochemistry:

• Optimize fixation conditions:

  • Test multiple fixation times (12-24 hours)

  • Compare different fixatives (10% NBF, zinc-based, alcohol-based)

  • Consider the impact of overfixation on epitope masking

• Enhance antigen retrieval:

  • Compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Test different retrieval times (10-30 minutes)

  • Try enzymatic retrieval for certain antigens

• Adjust antibody parameters:

  • Test serial dilutions (1/100 to 1/1000)

  • Extend incubation times (overnight at 4°C vs. 1 hour at room temperature)

  • Try different detection systems (polymer-based vs. avidin-biotin)

• Control for tissue variables:

  • Use positive control tissues processed identically

  • Consider tissue age and storage conditions

  • Evaluate impact of tissue thickness (4-5 μm optimal)

How can researchers quantitatively assess the binding kinetics of ERF010 antibody?

Bio-layer interferometry (BLI) provides a robust platform for determining binding kinetics:

• Immobilization approaches:

  • Immobilize the antibody onto anti-human IgG Fc capture (AHC) biosensors at 5 μg/ml

  • Alternatively, immobilize Fab fragments onto anti-human Fab-CH1 (FAB2G) biosensors at 25 μg/ml

• Antigen preparation:

  • Prepare serial dilutions of purified target protein, starting at 1000-2000 nM

  • Ensure protein quality through SEC purification

• Kinetic measurement settings:

  • Association step: 180-600 seconds

  • Dissociation step: 300-1200 seconds

  • Include reference sensors with buffer only for background subtraction

• Data analysis:

  • Use Octet System Data Analysis software for curve fitting

  • Apply 1:1 binding model to determine ka, kd, and KD values

  • Report confidence intervals for all kinetic parameters

A table summarizing typical binding parameters for high-affinity antibodies:

How should researchers design experiments to evaluate ERF010 antibody efficacy in inhibiting tumor growth in vivo?

A comprehensive in vivo efficacy assessment requires careful experimental design:

• Animal model selection:

  • Choose syngeneic models expressing the target receptor (similar to 4T1 or EMT6 models)

  • Consider patient-derived xenograft models for human target validation

  • For immunotherapy studies, use immunocompetent models

• Treatment regimen design:

  • Test multiple dose levels (e.g., 150 and 300 μg/mouse)

  • Establish dosing frequency (typically 2-3 times per week)

  • Begin treatment when tumors reach 50-100 mm³

• Endpoints and measurements:

  • Primary: Tumor volume measurements (2-3 times weekly)

  • Secondary: Tumor weight at study completion

  • Monitor body weight for toxicity assessment

  • Classify responses using clinical criteria (stable disease, partial response, complete response)

• Mechanism of action studies:

  • Perform immunophenotyping of tumor-infiltrating lymphocytes

  • Assess activated cytotoxic T lymphocytes (CTLs) (CD8+/GrB+)

  • Analyze immunosuppressive cell populations (TAMs, MDSCs)

What imaging techniques provide the most informative data on ERF010 antibody biodistribution?

Multiple imaging approaches can provide complementary information:

In Vivo Fluorescence Imaging:

• Label antibody with near-infrared fluorophores

• Allows longitudinal imaging in the same animal

• Limited by tissue depth penetration

• Best for subcutaneous models

PET/SPECT Imaging:

• Radiolabel antibody with ⁸⁹Zr (t½ = 78.4h) for PET or ¹¹¹In (t½ = 67.3h) for SPECT

• Provides whole-body biodistribution data

• Allows quantitative tissue uptake measurements

• Requires specialized facilities for radiochemistry

Ex Vivo Analyses:

• Immunofluorescence microscopy of tissue sections

• Multi-parameter analysis of co-localization with cell type markers

• High-resolution confocal imaging for cellular internalization studies

For optimal results, researchers should use a combination of these approaches, correlating in vivo biodistribution with ex vivo microscopic analyses of target engagement at the cellular level.