ARR7 Antibody

What is AR-V7 and why is it significant in prostate cancer research?

AR-V7 (Androgen Receptor Variant 7) is a clinically significant splice variant of the androgen receptor that has emerged as an important biomarker for guiding treatment decisions in castration-resistant prostate cancer (CRPC). AR-V7 is proposed to be ligand-independent and has been correlated with resistance to androgen deprivation therapy (ADT) .

The expression of AR-V7 is the most frequently identified disease-associated variant of the androgen receptor and is strongly correlated with CRPC development. Its detection in circulating tumor cells (CTCs) offers a non-invasive diagnostic approach that can help guide therapeutic decisions for patients with CRPC .

What is ARR7 in plant biology context and how does it function?

ARR7 (Arabidopsis Response Regulator 7) is a protein involved in cytokinin signal transduction in plants, specifically in Arabidopsis. It functions as an A-type response regulator and acts as a negative regulator of cytokinin signaling .

Research indicates that ARR7 requires phosphorylation at a specific aspartate residue (D85) to function properly in the cytokinin signaling pathway. When this residue is mutated (D85N), ARR7 loses its ability to be phosphorylated and consequently cannot mediate cytokinin responses. Experimental evidence shows that while overexpression of wild-type ARR7 decreases root-growth inhibition, callus formation, and cytokinin-inducible gene expression, overexpression of the non-phosphorylatable ARR7(D85N) mutant does not produce these phenotypes .

What techniques are most effective for validating AR-V7 antibody specificity?

Multiple complementary techniques should be employed to thoroughly validate AR-V7 antibody specificity:

• Western Blotting: Assess antibodies against protein lysates from cell lines with known AR-V7 status to confirm detection of a protein band at the expected size (~80 kDa) for AR-V7 .

• Immunocytostaining: Compare staining patterns in cell lines with known AR/AR-V7 status, examining for specific nuclear localization in AR-V7 positive cells and absence of signal in negative controls .

• Digital Droplet PCR (ddPCR): Validate AR-V7 status at the transcript level in cell lines used for antibody testing to confirm protein expression results .

• Cross-reactivity Assessment: Evaluate whether antibodies cross-react with full-length AR (AR-FL) or other proteins by testing in cell lines expressing different combinations of AR and AR-V7 .

• Positive and Negative Controls: Include controls in every experiment, particularly 22RV1 cells (AR-V7 positive) alongside AR-V7 negative cell lines to establish appropriate detection thresholds .

How do different AR-V7 antibodies compare in specificity and sensitivity?

A comprehensive evaluation of seven commercially available AR-V7 antibodies revealed significant variations in performance characteristics:

Notably, only the AG10008 antibody staining conveyed prognostic information and was associated with shorter progression-free survival in patients .

What critical factors influence AR-V7 antibody selection for circulating tumor cell detection?

When selecting antibodies for AR-V7 detection in CTCs, researchers should consider these crucial factors:

• Epitope Specificity: The antibody must specifically recognize the unique 16 amino acid sequence (EKFRVGNCKHLKMTRP) of AR-V7 without cross-reacting with full-length AR or other proteins .

• Signal-to-Noise Ratio: A high signal-to-noise ratio is essential for distinguishing true AR-V7 positive cells from background. Significant variation exists among commercially available antibodies .

• Nuclear Localization: Given that functional AR-V7 localizes to the nucleus, antibodies that produce clear nuclear staining patterns improve detection accuracy .

• Cross-Reactivity Profile: Minimal cross-reactivity with other proteins is crucial. Clone E308L emerged as having negligible cross-reactivity compared to other antibodies like AG1008, which showed strong reactions to proteins of sizes other than 80 kDa .

• Performance in Patient Samples: Validation in actual patient CTCs rather than just cell lines is essential, as cellular context can affect antibody binding characteristics .

Based on comprehensive testing using western blotting and immunocytostaining against cell lines with known AR/AR-V7 status, clone E308L emerged as the most reliable antibody, showing specific nuclear signals with optimal signal-to-noise ratio .

Why do different AR-V7 antibodies produce discordant staining results in some studies?

The discrepancies observed between different AR-V7 antibodies can be attributed to several technical and biological factors:

• Epitope Recognition Differences: Even antibodies targeting the same protein may recognize different epitopes. Some AR-V7 antibodies are developed against antigens containing not only the unique AR-V7 sequence but also parts of the DNA binding domain (DBD) shared with AR-FL, potentially affecting specificity .

• Antibody Generation Methods: Variations in antibody generation (monoclonal vs. polyclonal, host species, immunization protocols) result in different binding characteristics .

• Immunostaining Protocol Variations: Differences in fixation, antigen retrieval, blocking conditions, antibody concentrations, and detection systems influence staining patterns .

• Cross-Reactivity Profiles: Some antibodies exhibit significant cross-reactivity with non-AR-V7 proteins. For example, AG1008 produces strong reactions to proteins of sizes other than 80 kDa in all cell lines, including AR-V7 negative ones .

• Conformational Variation: AR-V7 protein conformation may vary in different cellular contexts or experimental conditions, affecting epitope accessibility .

These discrepancies have significant implications for clinical research, as demonstrated by a direct comparison of antibodies AG10008 and RM7 in tissue microarray samples, where despite similar positivity rates, there was only 7% agreement in staining intensities of positive cores .

What methodological approach should be used for detecting AR-V7 in circulating tumor cells?

A standardized methodological approach for AR-V7 detection in CTCs includes:

• CTC Enrichment: Isolate CTCs from blood samples using negative depletion of leukocytes to avoid potential antigen loss during positive selection .

• Sample Preparation: Spin cells onto glass slides (200 × g, 10 min) for immunocytostaining .

• Immunocytostaining Protocol:

  • Apply anti-AR-V7 antibody (preferably clone E308L)

  • Include anti-CD45 to exclude lymphocytes

  • Use nuclear stain (Hoechst) to confirm cellular identity

  • Define CTCs as CD45-negative, AR-V7-positive, and Hoechst-positive

• Positive Control Implementation: Run all patient CTC samples in parallel with 22RV1 cells (AR-V7 positive) to establish appropriate positivity thresholds. Research shows that weak but clear AR-V7 staining in 22RV1 was around an intensity value of 2000 (AR488 channel); events below this threshold should be considered AR-V7 negative .

• Signal Quantification: Use image analysis software to objectively measure signal intensities and apply consistent thresholds across samples .

This standardized approach ensures reliable AR-V7 detection in CTCs, which can inform treatment decisions for CRPC patients and enable research correlating AR-V7 subcellular localization with patient outcomes .

What is the significance of phosphorylation in ARR7 function and detection?

Phosphorylation plays a crucial role in ARR7 function in plant biology:

• Essential for Signal Transduction: Experimental evidence demonstrates that phosphorylation of ARR7 at the specific aspartate residue (D85) is necessary for its function in cytokinin signaling .

• Functional Validation: When this residue is mutated to asparagine (D85N), preventing phosphorylation, the protein loses its ability to function in cytokinin response pathways. While ectopic expression of wild-type ARR7 decreases root-growth inhibition, callus formation, and cytokinin-inducible gene expression, overexpression of ARR7(D85N) at similar levels does not produce these phenotypes .

• Protein Stability and Localization: Interestingly, the phosphorylation status does not affect the subcellular localization or half-life of ARR7. Both wild-type ARR7 and the ARR7(D85N) mutant localize to the nucleus and have similar half-lives in Arabidopsis mesophyll protoplasts .

• Mechanistic Implications: ARR7 functions within a multi-step two-component signaling (TCS) system utilizing histidyl-aspartidyl phosphorelays for cytokinin signal transduction in Arabidopsis. Phosphorylation acts as the key mechanism by which this signaling cascade transmits information .

For antibody-based detection methods, researchers must consider whether their detection approach is sensitive to the phosphorylation state of ARR7, especially when attempting to distinguish between active (phosphorylated) and inactive forms of the protein.

How can researchers rationally design antibodies to target specific epitopes in disordered proteins?

A cutting-edge approach to antibody design for targeting specific epitopes in disordered proteins involves:

• Sequence-Based Design: The method entails designing one or more complementary peptides specifically targeting a selected disordered epitope within a protein .

• Grafting Technique: After design, these complementary peptides are grafted onto an antibody scaffold to create a functional antibody with the desired specificity .

• Applications: This rational design approach has been successfully implemented to target three different disordered proteins and peptides associated with neurodegenerative and systemic misfolding diseases .

• Advantages: Compared to traditional immunization-based methods:

  • Enables targeting of weakly immunogenic epitopes

  • Provides greater control over epitope selection

  • Reduces time and cost, particularly when screening for specific epitopes is required

  • Allows targeting of virtually any chosen disordered epitope

This rational design method represents a significant advancement for researchers working with disordered proteins, potentially improving the specificity and reliability of antibodies used in detecting proteins like AR-V7 that contain disordered regions .

How can computational models support antibody design for optimal antigen specificity?

Advanced computational approaches are emerging to support antibody design with customized specificity profiles:

• Training Data Generation: Phage display experiments selecting antibodies against various combinations of ligands provide multiple training and test sets to build and assess computational models .

• Energy Function Optimization: The generation of new antibody sequences relies on optimizing energy functions associated with different binding modes. For cross-specific sequences, energy functions for desired ligands are minimized jointly. For specific sequences, energy functions for desired ligands are minimized while those for undesired ligands are maximized .

• Custom Specificity Profiles: Computational models can design novel antibody sequences with predefined binding profiles that are either:

  • Cross-specific: allowing interaction with several distinct ligands

  • Specific: enabling interaction with a single ligand while excluding others

• Experimental Validation: The efficacy of these computational predictions can be tested by synthesizing and experimentally validating antibody variants not present in the training set .

This computational approach represents a significant advancement in rational antibody design, potentially reducing the time and resources required for developing highly specific antibodies for research and diagnostic applications.

What evidence exists for AR-V7 expression in primary prostate cancer versus CRPC?

The presence of AR-V7 in primary prostate cancer versus castration-resistant prostate cancer (CRPC) has been a point of scientific debate:

These findings underscore the importance of antibody selection in AR-V7 detection and interpretation, while confirming that AR-V7 can indeed be present in primary prostate cancer. This has important implications for understanding disease progression and potentially identifying patients at higher risk of developing castration resistance .

How does AR-V7 detection in CTCs influence clinical decision-making for CRPC patients?

AR-V7 detection in circulating tumor cells has emerged as an important factor in guiding treatment decisions for CRPC patients:

• Biomarker for Treatment Selection: AR-V7 serves as a biomarker to guide treatment options for castration-resistant prostate cancer patients, particularly regarding the choice between AR-targeted therapies and alternative approaches .

• Non-invasive Assessment: Detection of AR-V7 in CTCs offers a non-invasive diagnostic avenue compared to tissue biopsies, allowing for repeated monitoring throughout treatment .

• Protein vs. Transcript Detection: While AR-V7 is detectable at the transcript level, protein detection in CTCs may provide additional information and clinical relevance. The ability to determine subcellular localization and potential co-localization with other proteins and cellular structures may offer insights into functional activity .

• Treatment Resistance Prediction: AR-V7 expression is correlated with resistance to androgen deprivation therapy, helping identify patients who might benefit from alternative treatment approaches .

• Standardization Needs: The choice of antibody and detection method significantly impacts results, highlighting the need for standardized protocols to ensure reliable clinical decision-making. Clone E308L has emerged as the most reliable antibody for this application .

Reliable AR-V7 detection in CTCs enables correlations between AR-V7 subcellular localization and patient outcomes, potentially improving treatment stratification and personalized medicine approaches for CRPC patients .