ATP7B Recombinant Monoclonal Antibody

What is ATP7B and why is it significant for research?

ATP7B is a transmembrane copper-transporting protein that plays a crucial role in copper homeostasis, primarily functioning in the liver to transport and regulate copper within the body. The significance of ATP7B in research stems from its central role in Wilson's disease pathophysiology, where mutations in the ATP7B gene lead to copper accumulation and subsequent tissue damage . Understanding ATP7B function provides insights into both normal copper metabolism and pathological states, making it an important target for researchers studying metal transport disorders, liver pathologies, and neurodegenerative conditions .

How are ATP7B recombinant monoclonal antibodies produced?

ATP7B recombinant monoclonal antibodies are generated through sophisticated in vitro processes using synthetic genes. The methodology involves:

• Isolation of ATP7B antibody genes from B cells of immunoreactive rabbits

• Amplification and cloning of these genes into appropriate phage vectors

• Introduction of vectors into mammalian cell lines (such as HEK293F)

• Production of functional antibodies in substantial quantities

• Purification from the culture supernatant through affinity chromatography

This recombinant approach offers advantages over traditional hybridoma-based methods, including better reproducibility, reduced batch-to-batch variation, and the ability to engineer specific properties of the antibody .

What are the primary research applications for ATP7B recombinant monoclonal antibodies?

ATP7B recombinant monoclonal antibodies can be utilized in multiple research applications:

Each application provides complementary information, allowing researchers to build a comprehensive understanding of ATP7B biology in their experimental systems .

How can I optimize immunofluorescence protocols for studying ATP7B trafficking in response to copper levels?

Optimizing immunofluorescence protocols for studying ATP7B trafficking requires careful consideration of several methodological factors:

• Cell fixation method selection: For preserving ATP7B localization during copper-induced trafficking, 4% formaldehyde fixation is recommended with minimal permeabilization time to prevent artificial redistribution of the protein .

• Copper treatment optimization:

  • For acute responses: Treat cells with 10-200 μM CuCl₂ for 1-4 hours

  • For chronic effects: Use 5-20 μM CuCl₂ for 12-48 hours

  • Always include appropriate controls with chelators (like D-penicillamine)

• Co-localization studies: Combine ATP7B antibody (dilution 1:100) with markers for different cellular compartments:

  • TGN markers (TGN46, Golgin97) for steady-state localization

  • Lysosomal markers (LAMP1) for degradation pathways

  • Vesicular markers (Rab7, Rab11) for trafficking routes

• Quantification approaches: Implement Pearson's correlation coefficient or Manders' overlap coefficient for objective assessment of co-localization under different copper conditions .

The trafficking dynamics can be significantly affected by ATP7B mutations, as demonstrated in studies where mutants like p.L168P and p.S1423N showed impaired intracellular trafficking despite normal mRNA expression .

What controls are critical when assessing ATP7B expression in Wilson's disease models?

When assessing ATP7B expression in Wilson's disease models, the following controls are essential for rigorous scientific investigation:

• Antibody validation controls:

  • ATP7B knockout cells as negative controls to confirm antibody specificity

  • Wild-type ATP7B overexpression systems as positive controls

  • Peptide competition assays to verify epitope specificity

• Expression level controls:

  • Housekeeping protein controls (HSC70 has been validated) for normalization

  • mRNA quantification via RT-qPCR to distinguish transcriptional vs. post-transcriptional effects

• Functional controls:

  • Copper exposure assays (copper concentrations causing 50% viability reduction)

  • Intracellular copper accumulation measurements (47.9 ± 8% reduction observed in ATP7B mutants)

  • Protein trafficking assessment at different temperatures (30°C can increase stability of some mutants by factors of ≈1.2-1.4)

• Patient-derived sample considerations:

  • Age-matched controls are crucial (ATP7B expression varies developmentally)

  • Tissue-specific controls (liver vs. brain expression patterns differ significantly)

Implementing these controls helps differentiate between true ATP7B deficiency states and experimental artifacts, particularly important when characterizing novel ATP7B variants .

How can ATP7B peptide measurements be integrated into research workflows for Wilson's disease diagnosis?

Integration of ATP7B peptide measurements into research workflows for Wilson's disease diagnosis represents a significant methodological advancement with specific implementation considerations:

• Sample collection and preparation protocol:

  • Dried blood spot (DBS) samples provide sufficient material with minimal invasiveness

  • Process samples within 24 hours or store at -80°C to preserve protein integrity

  • Standardize extraction protocols to ensure reproducibility

• Analytical methodology:

  • Immunoaffinity enrichment followed by mass spectrometry analysis

  • Two ATP7B peptides (ATP7B 887 and ATP7B 1056) serve as quantitative surrogate markers

  • Both peptides provide excellent diagnostic performance (AUC of 0.98)

• Performance characteristics and integration with other diagnostic approaches:

  Diagnostic Parameter	ATP7B Peptide Analysis Value	Comparison to Traditional Methods
  Sensitivity	91.2%	Superior to genetic testing alone
  Specificity	98.1%	Comparable to combined biochemical tests
  PPV	98.0%	Higher than ceruloplasmin testing
  NPV	91.5%	Superior to genetic testing alone

• Special case handling:

  • For patients with normal ceruloplasmin concentrations (>20 mg/dL): ATP7B peptide analysis identified 87.5% (14/16) of confirmed Wilson's disease patients

  • For patients with ambiguous genetic results: 94% were correctly identified as ATP7B deficient

This approach complements the Leipzig scoring system, providing a more accessible and potentially earlier diagnostic capability, especially valuable for pediatric patients where liver biopsy represents a significant risk .

How do I address discrepancies between ATP7B mRNA expression and protein detection in experimental systems?

Addressing discrepancies between ATP7B mRNA and protein levels requires systematic investigation of several potential mechanisms:

• Post-transcriptional regulation assessment:

  • Examine protein stability through cycloheximide chase experiments

  • Compare protein half-life between wild-type and variant ATP7B (variants like p.L168P show only 34.3 ± 8% protein expression despite normal mRNA levels)

  • Test temperature sensitivity (30°C incubation can increase stability of some mutants)

• Methodological considerations:

  • Ensure antibody epitope accessibility isn't affected by protein conformational changes

  • Try multiple antibodies targeting different domains of ATP7B

  • Consider native vs. denaturing conditions for protein analysis

• Evaluation of protein quality control mechanisms:

  • Assess proteasomal degradation through inhibitors (MG132)

  • Investigate autophagy involvement using bafilomycin A1

  • Analyze interaction with molecular chaperones (Hsp70, Hsp90)

• Systematic approach to troubleshooting:

  Observation	Potential Cause	Experimental Approach
  Normal mRNA with low protein	Enhanced degradation	Proteasome/autophagy inhibitors
  Normal mRNA with mislocalized protein	Trafficking defect	Immunofluorescence with organelle markers
  Protein detected only in certain compartments	Domain-specific antibody accessibility	Multiple antibodies targeting different regions
  Variable results between experiments	Environmental factors affecting stability	Standardize temperature, pH, copper levels

When interpreting such discrepancies, remember that they often reflect genuine biological phenomena rather than technical issues, as demonstrated in studies where ATP7B mutations resulted in normal transcription but defective protein expression or function .

What factors influence the reproducibility of ATP7B antibody performance across different research applications?

Multiple factors can influence ATP7B antibody reproducibility across research applications, requiring careful methodological consideration:

• Antibody source and production factors:

  • Recombinant monoclonal antibodies offer superior reproducibility compared to polyclonal antibodies

  • Expression systems affect glycosylation patterns (HEK293F cells provide mammalian-type modifications)

  • Clone selection is critical (reported clones include 24A4 for research applications)

• Sample preparation considerations:

  • ATP7B is a large transmembrane protein (157 kDa) requiring optimized extraction methods

  • Membrane protein solubilization conditions significantly impact epitope accessibility

  • Fixation methods for microscopy applications can mask or alter epitopes

• Application-specific optimization requirements:

  Application	Critical Factors	Optimization Approach
  Western Blot	Denaturation conditions	Test multiple buffer systems and temperatures
  Immunofluorescence	Fixation/permeabilization	Compare paraformaldehyde, methanol, and acetone fixation
  Flow Cytometry	Cell preparation	Test cell-specific permeabilization conditions
  IHC	Antigen retrieval	Compare heat-induced vs. enzymatic methods

• Environmental and experimental variables:

  • Copper levels significantly alter ATP7B trafficking and conformation

  • Temperature affects protein stability (particularly relevant for mutant variants)

  • Cell confluence and passage number impact expression levels

Maintaining detailed records of these variables and implementing standardized protocols across experiments is essential for achieving reproducible results with ATP7B antibodies .

How can ATP7B peptide analysis contribute to understanding variant pathogenicity in Wilson's disease research?

ATP7B peptide analysis offers innovative approaches to understanding variant pathogenicity through direct protein quantification:

• Functional classification of variants of uncertain significance (VUS):

  • Direct measurement of ATP7B peptides can differentiate between benign polymorphisms and pathogenic variants

  • Particularly valuable for novel variants where computational prediction may be unreliable

  • Quantitative correlation between peptide levels and disease severity provides functional insights

• Integration with genetic data:

  • ATP7B peptide analysis identified 94% of Wilson's disease patients with ambiguous genetic results

  • Provides complementary data to resolve cases where genomic information alone is insufficient

  • Helps distinguish compound heterozygotes from simple carriers

• Correlation with phenotypic manifestations:

  • ATP7B peptide levels in patients with normal ceruloplasmin (>20 mg/dL) showed 87.5% diagnostic accuracy

  • Offers quantitative metrics for genotype-phenotype correlation studies

  • Supplements Leipzig scoring system for more comprehensive disease characterization

• Methodological advantages for variant characterization:

  Traditional Approach	ATP7B Peptide Analysis Advantage	Research Impact
  Cell models with overexpressed variants	Direct measurement from patient samples	More physiologically relevant data
  Computational prediction of variant effects	Quantitative protein measurement	Empirical evidence of functional impact
  Binary classification (pathogenic/benign)	Quantitative scale of protein deficiency	Better reflects disease spectrum
  Requires extensive functional studies	Rapid assessment from DBS samples	Accelerates research timelines

This approach represents a significant advancement over traditional methods by providing direct measurement of the functional consequence of genetic variants, allowing researchers to establish more reliable genotype-phenotype correlations in Wilson's disease .

What methodological approaches can be used to study ATP7B trafficking dynamics in response to copper levels?

Studying ATP7B trafficking dynamics requires sophisticated methodological approaches to capture the protein's movement in response to changing copper concentrations:

• Live-cell imaging techniques:

  • ATP7B-GFP fusion protein expression systems for real-time visualization

  • Spinning disk confocal microscopy for rapid acquisition with minimal photobleaching

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility rates between compartments

  • Time-lapse imaging with defined copper exposure protocols (10-50 μM CuCl₂)

• Compartment-specific markers for co-localization studies:

  • TGN markers (TGN46, Syntaxin 6) for baseline localization

  • Vesicular markers (Rab7, Rab11, VAMP7) for trafficking intermediates

  • Apical membrane markers (MRP2) for polarized cells

  • Lysosomal markers (LAMP1) for degradation monitoring

• Quantitative analysis frameworks:

  • Automated image analysis using ImageJ/FIJI with JACoP plugin for co-localization

  • Tracking algorithms to follow ATP7B-positive vesicles

  • Intensity correlation analysis between ATP7B and compartment markers

  • 3D reconstruction of z-stack images for complete spatial representation

• Experimental design considerations:

  Research Question	Methodological Approach	Analytical Metrics
  Baseline distribution	Steady-state imaging in low copper	Pearson's correlation with TGN markers
  Kinetics of redistribution	Time-course after copper addition	Velocity of vesicle movement (μm/min)
  Recycling pathways	Copper chelation following redistribution	Recovery rate to baseline localization
  Trafficking defects in mutants	Comparison with wild-type	Quantitative differences in localization patterns

These approaches have revealed that ATP7B mutants associated with Wilson's disease often display impaired trafficking responses to copper, contributing to disease pathogenesis despite normal mRNA expression .

How can ATP7B antibodies be utilized in developing screening methods for Wilson's disease therapeutics?

ATP7B antibodies offer powerful tools for developing screening platforms to identify and evaluate potential Wilson's disease therapeutics:

• High-content screening approaches:

  • Automated immunofluorescence imaging of ATP7B localization in response to drug candidates

  • Quantitative assessment of trafficking restoration in disease models

  • Multiplexed readouts combining ATP7B localization with copper sensors

  • Cell-based assays using ATP7B-deficient cell lines (HepG2 KO cells expressing mutant ATP7B)

• Target engagement validation:

  • Co-immunoprecipitation studies to identify compounds that modulate ATP7B interactions

  • Thermal shift assays to detect stabilization of mutant ATP7B proteins

  • Surface plasmon resonance to measure direct binding of therapeutic candidates

  • CETSA (Cellular Thermal Shift Assay) for in-cell target engagement

• Functional rescue assessment:

  • Copper transport activity in polarized cell systems

  • ATP7B peptide quantification before and after treatment

  • Intracellular copper distribution using specialized probes

  • Cell viability under copper challenge (demonstrated to detect functional differences in ATP7B mutants)

• Therapeutic screening paradigms:

  Therapeutic Approach	Screening Method	Evaluation Metrics
  Protein stabilizers	Western blot quantification	Increase in ATP7B protein levels (p.L168P mutants show only 34.3±8% expression)
  Trafficking enhancers	Immunofluorescence localization	Restoration of copper-responsive trafficking
  Copper chelators	MTT viability assay under copper challenge	Protection from copper toxicity
  Gene therapy vectors	ATP7B peptide quantification	Restoration toward normal ATP7B levels

These methodologies enable rational therapeutic development by providing direct assessment of drug effects on ATP7B function and copper homeostasis, potentially leading to personalized approaches for specific ATP7B mutations .

How are ATP7B antibodies contributing to our understanding of the relationship between copper metabolism and neurodegenerative diseases?

ATP7B antibodies are facilitating groundbreaking research into the connections between copper dysregulation and neurodegenerative conditions:

• Brain-specific ATP7B expression patterns:

  • ATP7B isoform 2 is expressed in brain but not in liver, suggesting tissue-specific functions

  • Immunohistochemistry with ATP7B antibodies reveals regional distribution in the brain

  • Different expression patterns in neurons versus glial cells provide insights into cell-specific copper handling

• Copper homeostasis in neurological conditions:

  • ATP7B antibodies enable detection of subtle changes in copper transport proteins in brain tissues

  • Expression patterns can be compared between control and disease samples (Alzheimer's, Parkinson's)

  • Co-localization studies with disease-specific protein aggregates (β-amyloid, α-synuclein)

• Mechanistic investigations:

  • Determination of ATP7B subcellular localization in neuronal models

  • Assessment of ATP7B modifications (phosphorylation, ubiquitination) in response to oxidative stress

  • Evaluation of copper-dependent protein interactions in neural cell types

• Methodological applications in neurodegenerative research:

  Research Question	ATP7B Antibody Application	Potential Insight
  Regional copper distribution	IHC mapping of ATP7B in brain regions	Vulnerability patterns in neurodegeneration
  Copper handling in neural cell types	IF co-staining with neural markers	Cell-specific copper processing mechanisms
  Response to copper dyshomeostasis	Western blot of ATP7B in models	Adaptive mechanisms during pathological states
  Copper-protein interactions	IP-MS with ATP7B antibodies	Novel interaction partners in neural cells

This research direction is particularly significant as it bridges the established role of ATP7B in Wilson's disease with broader implications for neurological conditions where metal dyshomeostasis may contribute to pathogenesis .

What methodological considerations are important when developing quantitative assays for ATP7B in clinical samples?

Developing reliable quantitative assays for ATP7B in clinical samples requires careful attention to several critical methodological factors:

• Sample type selection and optimization:

  • Dried blood spots (DBS) provide practical advantages for clinical collection

  • Liver biopsy samples offer direct assessment of the primary ATP7B expression site

  • Peripheral blood mononuclear cells may serve as accessible surrogates

  • Sample stability conditions must be validated (storage at -80°C is recommended)

• Analytical methodology selection:

  • Immunoaffinity enrichment coupled with mass spectrometry offers superior specificity

  • Two validated ATP7B peptides (ATP7B 887 and ATP7B 1056) serve as quantitative markers

  • Both peptides demonstrate excellent diagnostic performance (AUC of 0.98)

  • Specific peptide sequences must be selected to avoid regions affected by common mutations

• Assay performance optimization:

  • Sensitivity must be sufficient to detect low-abundance ATP7B (91.2% sensitivity achieved)

  • Specificity requirements are stringent for diagnostic applications (98.1% achieved)

  • Reference ranges must be established across diverse populations

  • Quality control samples must represent the full analytical range

• Validation considerations for clinical implementation:

  Validation Parameter	Recommended Approach	Achievement Benchmark
  Analytical specificity	Test against related P-type ATPases	Demonstrate absence of cross-reactivity
  Analytical sensitivity	Establish LLOQ in clinical matrices	Detect ≤50% reductions in ATP7B levels
  Clinical validation	Compare against Leipzig scoring system	Demonstrate ≥90% concordance with confirmed cases
  Reproducibility	Inter-laboratory comparison studies	CV ≤20% across testing sites

These quantitative assays represent a significant advancement over traditional biochemical and genetic testing approaches, potentially enabling earlier diagnosis, particularly in pediatric patients where clinical presentation may be ambiguous .