AOC4 Antibody

What is AC4 and why are antibodies against it important in research?

AC4 (adenylate cyclase 4) is encoded by the ADCY4 gene and functions in GPCR signaling pathways and intracellular signal transduction. The human version has a canonical length of 1077 amino acid residues and a protein mass of 119.8 kilodaltons, with two identified isoforms. Anti-AC4 antibodies are crucial research tools that enable scientists to detect and measure AC4 antigen in biological samples, providing insights into cellular signaling mechanisms and pathway regulation. These antibodies have become fundamental in understanding the role of adenylate cyclase in various physiological and pathological processes .

Where is AC4 primarily expressed and localized within cells?

AC4 is widely expressed across many tissue types, making it relevant for diverse research applications. At the subcellular level, AC4 is primarily localized in the cell membrane and cytoplasm. This dual localization pattern reflects its functional role in transmembrane signaling, where it serves as a critical component in the conversion of extracellular signals to intracellular responses. Understanding this expression pattern is essential when designing experiments to study AC4's role in specific tissue contexts or subcellular compartments .

What are the primary applications for AC4 antibodies in research?

Anti-AC4 antibodies are versatile research tools with multiple validated applications including ELISA, Flow Cytometry, Western Blot, Immunoprecipitation, and Immunohistochemistry. Each application provides different insights: ELISA allows quantitative detection in solution, Flow Cytometry enables cellular-level detection, Western Blot confirms specificity and molecular weight, Immunoprecipitation isolates native protein complexes, and Immunohistochemistry reveals tissue distribution patterns. This methodological diversity makes AC4 antibodies valuable across various research domains from basic science to translational medicine .

What distinguishes AOCs from traditional antibodies and what are their research applications?

AOCs represent a sophisticated class of therapeutic molecules that combine the specificity of monoclonal antibodies with the precision of oligonucleotides. Unlike traditional antibodies that simply bind targets, AOCs deliver oligonucleotides (such as siRNAs) to specific tissues. This enables targeted genetic intervention through mechanisms like mRNA degradation. For researchers, AOCs offer unprecedented opportunities to study gene function in specific cell populations without the need for genetic manipulation of the entire organism. The hybrid nature of these molecules allows for the exploration of both protein-protein interactions and nucleic acid-mediated effects within a single experimental system .

How is the drug-to-antibody ratio (DAR) determined for AOCs, and why is this parameter critical?

The drug-to-antibody ratio (DAR) represents a critical quality attribute for AOCs that directly impacts therapeutic efficacy and pharmacokinetics. Traditional determination methods often require sample simplification, but novel approaches using native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS) now allow direct measurement through proteoform peak integration. This methodology involves truncation of Orbitrap's unreduced time-domain ion signals before mass spectra generation, providing DAR distribution and average values with less than 10% error. For researchers, precise DAR determination is essential for standardizing experiments, ensuring batch-to-batch consistency, and accurately interpreting dosage-dependent biological effects .

What are the key components of an optimized AOC, and how do they contribute to research applications?

An optimized AOC consists of three critical components, each carefully engineered for maximum efficacy:

• Monoclonal antibody (e.g., TfR1 mAb): Selected for target specificity and optimized through epitope selection and engineering to remove effector functions that might cause unwanted immunological effects. The antibody component determines tissue targeting and cellular uptake efficiency.

• Linker system: Non-cleavable structures enhanced for safety and durability, with carefully optimized oligonucleotide-to-antibody ratios that balance delivery efficiency with molecular stability.

• Oligonucleotide payload (e.g., siRNA): Engineered for stability against lysosomal enzymes, with sequences selected for potency and specificity while minimizing off-target effects.

Understanding these components is essential for researchers designing studies with AOCs, as each element influences experimental outcomes from cellular uptake to genetic silencing efficiency .

How can researchers validate the specificity of AC4 antibodies in their experimental systems?

Validating antibody specificity requires a multi-faceted approach beyond manufacturer specifications. Researchers should implement:

• Positive and negative control tissues/cells with known AC4 expression profiles

• siRNA knockdown or CRISPR knockout validation to confirm signal reduction

• Peptide competition assays to verify epitope specificity

• Multi-antibody validation using antibodies against different AC4 epitopes

• Western blot analysis confirming bands at the expected molecular weight (119.8 kDa for canonical AC4)

Additionally, researchers should consider isoform specificity, as two human AC4 isoforms have been identified. Validation parameters should be reported in publications to enhance reproducibility across research groups .

What analytical techniques provide the most comprehensive characterization of AOCs?

Comprehensive AOC characterization requires a multi-level analytical approach. Native mass spectrometry (MS) methodologies, particularly when hyphenated to ion mobility (IM-MS), provide exceptional insight by delivering multiple critical quality attributes in a single analysis. This approach gives researchers a direct snapshot of AOC homogeneity/heterogeneity without extensive data interpretation. For more detailed analysis, researchers should employ:

• Native size-exclusion chromatography coupled with Orbitrap FTMS

• Proteoform-level mass spectral peak integration

• Drug-to-antibody ratio (DAR) distribution analysis

• Conformational assessment via ion mobility MS

These methods can be applied to whole antibody conjugates or at the subunit level, with the advantage that sample purification or simplification procedures (like deglycosylation) can often be omitted, streamlining analytical workflows .

How can researchers optimize the specificity and potency of siRNAs in AOC development?

Developing highly specific and potent siRNAs for AOCs requires systematic optimization across multiple parameters:

• Screening methodology: Test candidate siRNAs in relevant donor cells (e.g., FSHD patient-derived myotubes for DUX4-targeting AOCs)

• Potency evaluation: Determine IC50 values and maximum inhibition (Emax) percentages using dose-response curves

• Off-target analysis: Conduct comprehensive RNA-seq to identify potential off-target effects

• Sequence modifications: Introduce chemical modifications to enhance stability against nucleases while maintaining RNAi activity

• Target validation: Verify downregulation of target-regulated genes (e.g., DUX4-regulated genes like KHDC1L, LEUTX, MBD3L2 for DUX4-targeting AOCs)

The data table below illustrates how potency can vary among siRNA candidates tested in patient-derived cells:

This methodical approach ensures selection of siRNA sequences with optimal efficacy and minimal off-target effects .

How should researchers interpret differences in antibody binding between monomeric and oligomeric forms of target proteins?

Interpreting differential antibody binding between monomeric and oligomeric protein forms requires careful consideration of structural biology principles. For example, studies with aquaporin-4 (AQP4) antibodies reveal significantly greater binding to the M23-AQP4 isoform (which forms orthogonal arrays) compared to the M1-AQP4 isoform (which does not). This demonstrates how protein quaternary structure dramatically affects antibody accessibility and binding kinetics. When investigating AC4 or similar targets, researchers should:

• Characterize the oligomeric state of their target under experimental conditions

• Determine if the antibody epitope might be masked or exposed differently in various quaternary structures

• Consider using multiple antibodies recognizing different epitopes

• Employ native gel electrophoresis to confirm oligomeric states alongside immunodetection

• Interpret fluorescence patterns (smooth vs. punctate) as potential indicators of protein organization

Understanding these structural considerations is crucial for accurate data interpretation and can explain seemingly contradictory results between different experimental approaches .

What strategies can researchers employ when antibodies show unexpected cross-reactivity or background in their experimental system?

Addressing unexpected cross-reactivity requires systematic investigation and methodological refinement:

• Blocking optimization: Test different blocking agents (BSA, milk, serum) and concentrations to reduce nonspecific binding

• Antibody titration: Determine the minimum concentration needed for specific signal detection to improve signal-to-noise ratio

• Sample preparation modifications: Adjust fixation methods, permeabilization agents, or extraction buffers to better preserve epitopes while reducing nonspecific binding

• Secondary antibody controls: Run controls with secondary antibody only to identify potential direct binding to the sample

• Pre-adsorption: Pre-incubate antibody with purified antigen or peptide competitors to validate specificity

• Alternative detection systems: Try different detection methods (fluorescent vs. chromogenic) or amplification systems to improve specificity

When troubleshooting, it's important to modify only one variable at a time and maintain detailed records of optimization experiments for reproducibility and reporting purposes .

How can researchers accurately assess AOC pharmacokinetics and biodistribution in pre-clinical models?

Accurate assessment of AOC pharmacokinetics and biodistribution requires sophisticated analytical approaches beyond traditional ligand-binding assays (LBAs). Researchers should implement:

• LBA-LC-HRMS (high-resolution mass spectrometry): This hybrid approach combines immunocapture with chromatographic separation and intact mass detection, revealing potential biotransformation products not detected by traditional methods

• Intact quantification methods: Develop methods with appropriate linear dynamic ranges (e.g., 1-10 μg/mL) using minimal sample volumes

• Comparative analysis: Run parallel assays using both traditional LBA-LC-MRM and intact quantification to obtain complementary data

• Tissue distribution studies: Analyze multiple tissues to understand AOC targeting efficiency and off-target accumulation

• Time-course experiments: Collect samples at multiple timepoints to characterize absorption, distribution, metabolism, and excretion profiles

This multi-faceted analytical strategy provides deeper insights into AOC behavior in vivo, which is critical for optimizing dosing regimens and predicting clinical pharmacokinetics .

How might developments in mass spectrometry further enhance AOC characterization and quality control?

The evolution of mass spectrometry techniques continues to transform AOC characterization. Future developments will likely include:

• Higher resolution native MS approaches: Enabling better distinction between closely related species in complex conjugate mixtures

• Automated transient signal processing: Integration of artificial intelligence algorithms to optimize peak integration and improve DAR estimation accuracy

• Hyphenated techniques: Further development of multi-dimensional approaches combining ion mobility, native chromatography, and high-resolution MS

• Miniaturized sample preparation: Microfluidic systems integrated with MS for higher throughput and reduced sample consumption

• Real-time monitoring capabilities: Adaptation of MS techniques for in-process monitoring during AOC manufacturing

These advancements will enable more comprehensive characterization with less sample manipulation, allowing researchers to maintain native conformations and obtain more relevant structural and functional information about their AOC constructs .

What are the emerging applications of AOCs beyond current therapeutic targets?

While current AOC development focuses primarily on muscular dystrophies like FSHD, the platform's versatility suggests numerous emerging applications:

• Neurological disorders: Delivering oligonucleotides across the blood-brain barrier by leveraging transferrin receptor targeting

• Autoimmune conditions: Targeting pathogenic RNA species in immune cells with cell-type specific antibodies

• Metabolic diseases: Modulating gene expression in hepatocytes or adipocytes through tissue-specific delivery

• Cancer therapeutics: Combining the precision of antibody targeting with gene silencing to address oncogenic drivers

• Infectious diseases: Targeting viral RNA while simultaneously engaging host immune responses

Researchers should consider these emerging applications when designing AOCs, potentially developing modular platforms where antibody components can be exchanged to redirect the same oligonucleotide payload to different tissue targets .