ET3 Antibody

What are the primary targets of ET3 antibodies in research?

ET3 antibodies target two distinct proteins in research contexts:

• 4R Tau Isoform: The ET3 antibody specifically recognizes the amino acid sequence KVQIINKKLDLSNVQSK found in exon 10 of human tau. This antibody is critical for distinguishing 4-repeat (4R) tau isoforms from 3-repeat (3R) tau isoforms in neurodegenerative disease research .

• Endothelin-3 (EDN3): ET3 antibodies also target endothelin-3, a 21-amino acid peptide involved in cell proliferation, axon guidance, and other biological functions. The human version of ET3 has a canonical length of 238 amino acid residues and a protein mass of 25.5 kilodaltons .

How are ET3 antibodies typically generated for tau research?

ET3 tau-specific monoclonal antibodies are generated through a well-established immunization protocol:

• Tau-deficient (-/-) mice are immunized with a synthetic peptide corresponding to the amino acid sequence KVQIINKKLDLSNVQSK from exon 10 of human tau

• The peptide is cross-linked with glutaraldehyde and injected intraperitoneally (0.2 ml/injection with 1-2 mg/ml solution)

• Serum titers are determined by ELISA using biotinylated peptides and Western blot with recombinant tau fusion proteins

• Spleen cells from mice with highest antibody titers are fused with myeloma cells (NSO cells) using polyethylene glycol

• Fusion products are cultured in selection medium containing hypoxanthine-aminopterin-thymidine (HAT)

• Positive clones are identified by both ELISA and Western blot assays

This process has yielded three highly specific 4R tau monoclonal antibodies: ET1, ET2 (both IgG1) and ET3 (IgG2b) .

What are the optimal protocols for ET3 antibody application in immunohistochemistry?

For optimal 4R tau detection in immunohistochemistry using ET3 antibody:

• Fix specimens appropriately (paraformaldehyde for cell cultures, formalin for tissue sections)

• For tissue sections: perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

• Block with 5% milk in TBS for 1 hour at room temperature

• Apply ET3 antibody at a 1:100-1:200 dilution and incubate overnight at 4°C

• Use appropriate secondary antibodies (for ET3 IgG2b, use anti-mouse IgG2B Alexaflour 488)

• For dual labeling with RD3 (3R tau antibody), use different fluorophores for each secondary antibody

• Mount specimens using appropriate mounting media (e.g., Fluoromount)

For endothelin-3 detection, a similar protocol can be used with the following modifications:

• Use the endothelin-3 specific antibody at 25 μg/mL for 3 hours at room temperature

• Counterstain with DAPI for nuclear visualization

How do the detection sensitivities compare between Western blot, ELISA, and immunohistochemistry for ET3 antibodies?

The choice of method depends on research questions:

• Western blot provides size verification of 4R tau isoforms but may miss conformational epitopes

• ELISA offers greater sensitivity for quantitative analysis

• Immunohistochemistry provides crucial spatial information but requires careful validation against controls

How can ET3 antibodies be used to differentiate tau isoform profiles across different tauopathies?

ET3 antibodies are instrumental in characterizing tau isoform signatures across tauopathies when used in combination with 3R tau-specific antibodies (RD3):

This distinct pattern of ET3 immunoreactivity helps researchers characterize and distinguish between tauopathies. In CTE specifically, the ratio of 4R:3R tau-positive cells can be calculated to evaluate disease progression, with early stages showing predominantly 4R tau that shifts toward increased 3R tau in later stages .

What are the methodological considerations when using ET3 antibodies to study tau propagation in cellular models?

When using ET3 antibodies to study tau propagation in cellular models:

• Cell Model Selection:

  • Engineered iPSC-derived neurons expressing 4R tau (such as the engineered iPSC lines described in result ) provide optimal models

  • N2a cells transiently transfected with 4R tau cDNA can be used for preliminary studies

• Seeding Protocol Optimization:

  • Pre-formed tau fibrils must be prepared with careful quality control

  • Concentration and timing of seed introduction must be standardized

  • Negative controls using non-4R tau expressing cells are essential

• Detection Methods:

  • Use ET3 (4R-specific) in combination with phospho-tau antibodies to distinguish newly formed aggregates

  • Quantify using high-content imaging or flow cytometry for objective measurement

  • Consider utilizing CRISPRi screens to identify modifiers of tau propagation

• Challenges and Solutions:

  • Background signal: Thorough blocking (5% milk in TBS) and extensive washing

  • Timing: Progressive sampling (24h, 48h, 72h, 1 week) to capture dynamic changes

  • Validation: Orthogonal techniques (biochemical fractionation plus ET3 Western blotting)

The engineered human iPSC model expressing 4R-tau provides a powerful platform to identify novel therapeutic strategies for 4R tauopathy, and has been used to identify over 500 genetic modifiers of tau-seeding-induced tau propagation .

How can researchers address specificity concerns when ET3 antibody results conflict with other tau isoform markers?

When ET3 antibody results conflict with other tau markers:

• Verify Antibody Specificity:

  • Perform validation with recombinant tau isoforms (all six isoforms)

  • Include phosphatase treatment to eliminate effects of phosphorylation on epitope recognition

  • Test with tau knockout models as negative controls

• Cross-Validation Approach:

  • Compare ET3 with alternative 4R tau antibodies

  • Use multiple approaches (Western blot, IHC, ELISA) to confirm findings

  • Apply quantitative RT-PCR to measure 4R tau mRNA levels

• Common Sources of Discrepancy:

  • Phosphorylation status affecting epitope accessibility

  • Protein conformation differences between techniques

  • Fixation artifacts in histological preparations

  • Cross-reactivity with other microtubule-associated proteins

• Resolution Strategy:

  • Dephosphorylate samples with phosphatase to standardize phospho-status

  • Test multiple epitope exposure techniques

  • Validate using genetic controls (tau knockout and 4R tau-specific expression models)

What are the critical quality control parameters for validating new batches of ET3 antibodies?

For rigorous quality control of new ET3 antibody batches:

Quality control testing should include:

• Direct ELISA using cell lysates from COS-7 cells transiently transfected with tau

• Western blot analysis using recombinant tau proteins and brain homogenates

• Immunocytochemistry on cells expressing distinct tau isoforms

• Phosphatase treatment to ensure epitope recognition is not phosphorylation-dependent

How can ET3/endothelin-3 antibodies be utilized in cancer research models?

ET3/endothelin-3 antibodies have emerging applications in cancer research:

• Diagnostic Applications:

  • IHC studies have shown that 100% of melanoma patient lymph node biopsies were positive for RB49 (an antibody targeting activated endothelin B receptor, which binds ET3)

  • ET3 antibodies can be used to characterize endothelin receptor expression patterns in various tumor types

• Therapeutic Development:

  • ET3 antibodies can help evaluate endothelin pathway targeting in experimental models

  • They can be used to monitor the efficacy of endothelin receptor antagonists

  • Antibody-drug conjugates targeting the endothelin pathway show promise, as demonstrated by xiRB49-MMAE which showed high efficacy against melanoma cells and ET B+ xenograft tumor models

• Mechanistic Studies:

  • ET3 antibodies help elucidate the role of endothelin signaling in tumor progression

  • They can identify endothelin-producing cells within the tumor microenvironment

  • Measuring ET3 in LNCaP human prostate cancer cell line has revealed specific localization to cell secretion

The ET3/endothelin pathway represents a significant target in melanoma and other cancers, with ET receptor B+ tumors showing particular promise for targeted therapies.

How can ET3 antibodies be integrated with emerging technologies for advanced tau research?

Integration of ET3 antibodies with emerging technologies offers new research possibilities:

• High-Resolution Microscopy:

  • Super-resolution microscopy (STORM/STED) with ET3 antibodies can reveal nanoscale organization of 4R tau aggregates

  • Expansion microscopy can provide enhanced spatial resolution of tau pathology

  • Combining ET3 with other tau antibodies in multiplexed imaging enables comprehensive tau characterization

• Single-Cell Technologies:

  • ET3 antibodies can be adapted for CyTOF analysis to measure 4R tau in conjunction with other cellular markers

  • Single-cell sorting based on ET3 immunoreactivity followed by transcriptomics can identify cell-specific responses to tau pathology

• In vivo Applications:

  • Development of ET3-derived imaging agents for PET/SPECT to visualize 4R tau in living subjects

  • Modification of ET3 for blood-brain barrier penetration to enable therapeutic targeting

• Computational Integration:

  • Quantitative image analysis algorithms specific for ET3 staining patterns can automate detection

  • Machine learning approaches incorporating ET3 immunoreactivity patterns may improve tauopathy classification

• CRISPR-Based Approaches:

  • ET3 antibodies combined with CRISPR screens have already identified over 500 genetic modifiers of tau propagation

  • Further integration with base editing and epigenetic modulation technologies could reveal new therapeutic targets

What are the critical methodological considerations when using ET3 and RD3 antibodies to quantify tau isoform ratios in complex tissue samples?

For accurate quantification of tau isoform ratios using ET3 (4R) and RD3 (3R) antibodies:

• Sample Preparation Standardization:

  • Consistent fixation protocols (duration, fixative composition) are essential

  • Antigen retrieval methods must be optimized and standardized for both antibodies

  • Serial sections should be used for comparative analysis

• Quantification Approach:

  • Automated cell counting systems should establish clear positivity thresholds

  • Area measurement standardization (mm²) ensures comparable results between studies

  • For 4R/3R positive cell ratios, calculate the average number of 4R cells divided by the average number of 3R cells for each case

• Controls and Normalization:

  • Include known 3R-predominant and 4R-predominant cases as controls

  • Use of adjacent sections stained with a pan-tau antibody for total tau normalization

  • Age-correction is critical as tau isoform ratios change with aging

• Statistical Analysis:

  • Log transformation of 4R/3R ratios may be necessary to normalize for regression analysis

  • Multiple linear regression analysis should examine if 4R/3R ratios correlate with p-tau staining density independent of age and amyloid plaque score

  • Analysis of covariance (ANCOVA) can determine if age is implicated in the tau-containing neuron vs. astrocyte percentage results

• Technical Limitations to Address:

  • Phosphorylation can mask epitopes; dephosphorylation with phosphatase may be necessary

  • Background staining differences between antibodies require careful threshold setting

  • Cell-type specificity requires double-labeling with neuronal/glial markers

When applied correctly, this methodology has revealed that in conditions like CTE, neurons contain both 3R and 4R tau, while astrocytes contain exclusively 4R tau, with the 4R:3R ratio shifting toward 3R in advanced disease stages .