NDUFB3 Antibody, HRP conjugated

What is NDUFB3 and what is its role in mitochondrial function?

NDUFB3 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). While not directly involved in catalysis, it plays a crucial structural role in Complex I assembly and stability. Complex I facilitates electron transfer from NADH to the respiratory chain, with ubiquinone being the immediate electron acceptor . NDUFB3 is an integral component of subcomplex Iβ of the hydrophobic membrane arm, essential for proper assembly and function of the respiratory chain . The protein has a molecular weight of approximately 11 kDa and is encoded by the nuclear genome, making it subject to Mendelian inheritance patterns .

What methods can be used to validate NDUFB3 antibody specificity?

Validating NDUFB3 antibody specificity requires multiple complementary approaches:

• Western blot analysis:

  • Confirm the detection of a single band at the expected molecular weight (11 kDa)

  • Compare band patterns across multiple tissue types known to express NDUFB3 (heart, brain, liver, kidney)

  • Verify consistent detection across recommended dilution ranges (typically 1:500-1:2000 for polyclonal and 1:1000-1:8000 for monoclonal antibodies)

• Immunohistochemistry controls:

  • Include tissue sections with known high NDUFB3 expression (heart, liver) as positive controls

  • Employ primary antibody omission and isotype controls to confirm binding specificity

  • Compare staining patterns with published literature on mitochondrial protein distribution

• Advanced validation strategies:

  • Employ genetic models (knockdown/knockout systems) to confirm signal reduction/loss

  • Use multiple antibodies targeting different NDUFB3 epitopes to corroborate findings

  • Implement peptide competition assays to demonstrate epitope-specific binding

How does HRP conjugation benefit NDUFB3 antibody applications?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages for NDUFB3 detection:

• Enhanced experimental efficiency:

  • Eliminates the need for secondary antibody incubation and washing steps, reducing protocol time by 1-2 hours

  • Enables direct detection systems with simplified workflows, particularly valuable for high-throughput screening

  • Reduces potential cross-reactivity issues associated with secondary antibodies

• Signal optimization benefits:

  • Provides enzymatic signal amplification, enhancing detection sensitivity for low-abundance targets

  • Offers compatibility with multiple detection substrates (colorimetric, chemiluminescent, chemifluorescent)

  • Maintains longer-term stability compared to fluorophore conjugates, allowing extended storage periods

• Application versatility:

  • Particularly valuable for ELISA applications, enabling direct quantification of NDUFB3 levels

  • Useful for immunohistochemistry applications requiring sensitive detection with minimal background

  • Compatible with multiplexing when combined with other detection systems (e.g., alkaline phosphatase conjugates)

What are the key reactive species and tissue sources for NDUFB3 antibody applications?

Based on the search results, commercially available NDUFB3 antibodies demonstrate reactivity with:

• Species reactivity profile:

  • Human: Primary validated reactivity across multiple antibody products

  • Mouse: Confirmed reactivity for many polyclonal and some monoclonal antibodies

  • Rat: Verified reactivity primarily with polyclonal antibodies

• Optimal tissue sources for positive controls:

  • Heart tissue: Consistently shows the strongest NDUFB3 signals across species due to high mitochondrial content

  • Brain tissue: Reliable secondary option with moderate to strong expression levels

  • Liver and kidney: Also exhibit substantial NDUFB3 expression suitable for validation

• Cell line validation sources:

  • HeLa, HepG2, and A431 cells have been validated for NDUFB3 expression

  • Require higher protein loading (20-30 μg) compared to tissue samples (10-15 μg)

The NDUFB3 protein shows high conservation across mammals, explaining the cross-reactivity with multiple species. Researchers should verify specific reactivity claims for individual antibody products, as cross-reactivity can vary based on the epitope targeted and antibody format.

What are the recommended dilutions for NDUFB3 antibodies across different applications?

Optimal dilutions vary by antibody format, application, and sample type:

These dilutions should be optimized for each experimental system, considering:

• Sample type (tissue vs. cell lysate)

• Species source and protein abundance

• Detection method sensitivity

• Total protein loaded (typically 10-20 μg for tissue lysates)

How should samples be prepared for optimal NDUFB3 detection in Western blotting?

For reliable NDUFB3 detection in Western blotting, sample preparation must address its properties as a small (11 kDa) mitochondrial membrane protein:

• Optimal lysis protocol:

  • Use buffers containing ionic detergents (RIPA or modified RIPA) with protease inhibitor cocktails

  • For enhanced mitochondrial protein extraction, consider specialized mitochondrial isolation kits

  • Maintain cold conditions throughout extraction to prevent protein degradation

• Protein denaturation considerations:

  • Use reducing conditions (with β-mercaptoethanol or DTT) to break disulfide bonds

  • Heat samples at 95°C for 5 minutes to fully denature protein complexes

  • Avoid repeated freeze-thaw cycles that can degrade small proteins

• Gel separation optimization:

  • Use higher percentage gels (12-15% SDS-PAGE) for optimal separation of the low molecular weight NDUFB3

  • Consider gradient gels (4-20%) when analyzing multiple complex I components simultaneously

  • Load appropriate positive controls (heart tissue lysate demonstrates strongest signal)

• Transfer parameters:

  • Use PVDF membranes with 0.2 μm pore size (rather than 0.45 μm) to better retain small proteins

  • Optimize transfer conditions: shorter time (60-75 minutes) and lower voltage for small proteins

  • Include methanol (10-20%) in transfer buffer to facilitate membrane binding of hydrophobic proteins

• Blocking and antibody incubation:

  • 5% non-fat dry milk in TBST is consistently recommended as blocking/dilution buffer across multiple antibody products

  • Extended primary antibody incubation (overnight at 4°C) improves signal-to-noise ratio

What controls should be included when using NDUFB3 antibodies in experimental workflows?

A comprehensive control strategy ensures reliable NDUFB3 antibody results:

How do monoclonal and polyclonal NDUFB3 antibodies differ in research applications?

Monoclonal and polyclonal NDUFB3 antibodies present distinct advantages for specific research applications:

Application-specific considerations:

• For Western blotting: Both types work well when optimized; monoclonals provide more consistent results for longitudinal studies

• For fixed tissue IHC: Polyclonals may provide better signal in partially denatured samples

• For co-immunoprecipitation: Monoclonals may reduce background but polyclonals can capture more protein

• For detecting specific NDUFB3 variants: Epitope-specific monoclonals can distinguish wild-type from mutant forms if the epitope includes the mutation site

How can NDUFB3 antibodies be used to investigate Complex I assembly in mitochondrial disease models?

NDUFB3 antibodies provide valuable tools for studying Complex I assembly defects, particularly in disease models:

• Blue Native PAGE (BN-PAGE) combined with Western blotting:

  • This technique separates intact mitochondrial complexes without denaturation

  • NDUFB3 antibodies detect both fully assembled Complex I (~980 kDa) and assembly intermediates

  • In patients with NDUFB3 mutations, partially assembled Complex I intermediates of ~650 kDa are observed, representing accumulation of subcomplex Iβ assembly intermediates

  • This approach distinguishes between assembly defects and stability issues affecting Complex I

• Sequential immunoprecipitation strategies:

  • NDUFB3 antibodies can pull down intact complexes and interacting partners

  • Mass spectrometry analysis of co-precipitated proteins reveals successful or failed assembly partnerships

  • Comparison between control and disease models identifies the stage at which assembly is disrupted

  • This approach has helped establish NDUFB3's role in subcomplex Iβ of the hydrophobic membrane arm

• Immunofluorescence co-localization analysis:

  • NDUFB3 antibodies combined with antibodies against other Complex I subunits (NDUFA9, NDUFB8)

  • Co-localization coefficient analysis quantifies proper integration into the complex

  • Altered distribution patterns in mutant cells indicate specific assembly defects

  • Particularly valuable for analyzing patient-derived fibroblasts or muscle biopsies

• Time-course experiments in cellular models:

  • Pulse-chase experiments tracking newly synthesized NDUFB3 incorporation into Complex I

  • Reveals kinetic defects in assembly processes in disease models

  • Can assess effects of therapeutic interventions on assembly dynamics

What experimental approaches can correlate NDUFB3 expression with mitochondrial function?

Multiple methodologies can be integrated with NDUFB3 immunodetection to establish structure-function relationships:

• Combined biochemical and structural analysis:

  • NDUFB3 protein quantification by Western blot with antibodies of defined specificity

  • Parallel assessment of Complex I activity by spectrophotometric NADH:ubiquinone oxidoreductase assays

  • Correlation of protein levels with enzymatic function provides insight into threshold effects

  • The search results indicate that decreased steady-state levels of Complex I subunit proteins (NDUFB8 and NDUFA9) correlate with reduced Complex I activity in patients with NDUFB3 mutations

• Integrated cellular bioenergetics assessment:

  • Oxygen consumption rate (OCR) measurements using Seahorse analyzers

  • Membrane potential assessment using potentiometric dyes (TMRM, JC-1)

  • ATP production capacity under different substrate conditions

  • Correlation with NDUFB3 levels to establish functional consequences of altered expression

• Proteomics approaches:

  • Quantitative proteomics to measure stoichiometric changes in all Complex I subunits

  • Analysis of post-translational modifications affecting NDUFB3 stability or interactions

  • Interactome mapping to identify altered protein-protein interactions in disease states

  • These approaches have helped establish that NDUFB3 defects affect the entire Complex I assembly process

• Genetic manipulation experiments:

  • CRISPR/Cas9-mediated NDUFB3 mutation or deletion to model disease states

  • Rescue experiments with wild-type or mutant NDUFB3 constructs

  • Dose-dependent expression studies to determine critical thresholds for function

  • These approaches can help explain the phenotypic variability observed with identical mutations

How can researchers investigate the p.Trp22Arg NDUFB3 mutation's effects using antibody-based approaches?

The recurrent c.64T>C, p.Trp22Arg NDUFB3 mutation offers an important research model for mitochondrial disease. Antibody-based techniques provide valuable insights:

• Comparative protein expression analysis:

  • Western blotting with NDUFB3 antibodies quantifies protein levels in patient samples versus controls

  • Demonstrated reduced steady-state levels of not only NDUFB3 but also other Complex I components like NDUFB8 and NDUFA9

  • This suggests the mutation destabilizes the entire complex rather than just affecting the mutant protein

• Assembly defect characterization:

  • BN-PAGE analysis reveals decreased fully assembled Complex I in patient samples

  • The presence of approximately 650 kDa Complex I assembly intermediates indicates stalled assembly of the membrane arm

  • Immunoblotting with NDUFB8 reveals these partial assemblies, consistent with defects in subcomplex Iβ formation

• Structure-function investigation:

  • Immunoprecipitation of wild-type versus mutant NDUFB3 to analyze differential binding partners

  • Analysis of protein stability using cycloheximide chase experiments with Western blot detection

  • Subcellular fractionation to determine if the mutation affects mitochondrial targeting

• Genotype-phenotype correlation studies:

  • The search results reveal that patients with identical p.Trp22Arg mutations show variable clinical severity

  • This suggests additional genetic or environmental modifiers

  • Antibody-based proteomic approaches can help identify compensatory mechanisms in milder cases

• Therapeutic strategy assessment:

  • Monitoring NDUFB3 and Complex I levels following experimental therapies

  • Quantifying rescue of assembly defects as a primary endpoint

  • Correlation with functional recovery in cellular or animal models

What approaches can distinguish between reduced NDUFB3 expression and altered protein conformation?

Distinguishing between expression defects and conformational changes requires multiple complementary approaches:

• Epitope-specific antibody analysis:

  • Utilize multiple NDUFB3 antibodies targeting different epitopes

  • Differential detection patterns suggest conformational changes affecting epitope accessibility

  • Consistent reduction across antibodies suggests decreased expression

  • This approach is particularly valuable for assessing the effects of point mutations like p.Trp22Arg

• mRNA-protein correlation analysis:

  • Compare NDUFB3 mRNA levels (by RT-qPCR) with protein levels (by Western blot)

  • Discordance (normal mRNA with low protein) suggests post-transcriptional issues such as protein instability

  • Proportional reduction in both suggests transcriptional regulation or gene deletion

  • This helps determine at what level the defect manifests (transcription, translation, or post-translational)

• Protein stability assessment protocols:

  • Cycloheximide chase experiments with antibody detection to measure NDUFB3 half-life

  • Proteasome inhibition (MG132) with Western blot analysis to determine if degradation is enhanced

  • Pulse-chase studies to track protein maturation and incorporation into complexes

  • These approaches can reveal if mutations lead to increased protein turnover

• Structural analysis techniques combined with immunodetection:

  • Limited proteolysis with antibody detection reveals conformational differences that alter protease sensitivity

  • Detergent solubility profiling with immunoblotting detects aggregation propensity

  • Native versus denaturing gel electrophoresis comparison identifies conformational dependencies

  • These methods provide insight into how mutations might affect protein folding and complex assembly

What are common technical challenges when detecting NDUFB3 by Western blot?

Researchers frequently encounter technical issues when working with NDUFB3 in Western blot applications:

• Low molecular weight detection challenges:

  • NDUFB3's small size (11 kDa) can lead to it running off standard gels or poor retention during transfer

  • Solution: Use higher percentage gels (15-20%) or gradient gels (4-20%)

  • Optimize transfer conditions specifically for small proteins (shorter time, lower voltage, cold transfer buffer)

  • Use 0.2 μm pore size PVDF membranes rather than 0.45 μm or nitrocellulose

• Signal intensity optimization:

  • Low abundance in some samples can result in weak signals

  • Solutions: Increase protein loading (20-30 μg for cell lines, 10-15 μg for heart tissue)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use signal enhancement systems (high-sensitivity ECL substrates)

  • Consider using HRP-conjugated primary antibodies to eliminate signal loss from secondary antibody steps

• Specificity concerns:

  • Multiple bands or high background can complicate interpretation

  • Solutions: Validate specificity using heart tissue as positive control (highest expression)

  • Optimize blocking conditions (5% NFDM/TBST is consistently recommended)

  • Titrate antibody concentration carefully (excessive antibody increases background)

  • Consider monoclonal antibodies for highest specificity

• Sample-dependent variations:

  • Expression levels vary dramatically across tissue types

  • Solutions: Include tissue-matched controls

  • Adjust loading volume based on expected expression level

  • Use mitochondrial markers rather than typical housekeeping proteins for normalization

How can researchers optimize NDUFB3 immunohistochemistry protocols?

Achieving specific NDUFB3 detection in tissues requires systematic optimization:

• Antigen retrieval optimization:

  • The search results indicate TE buffer pH 9.0 is recommended for some NDUFB3 antibodies in IHC applications, with citrate buffer pH 6.0 as an alternative

  • Test multiple retrieval methods (heat-induced versus enzyme-based) with controlled temperature and duration

  • Optimize based on tissue type and fixation method used

• Signal amplification considerations:

  • For low-abundance detection, consider tyramide signal amplification (TSA)

  • Polymer detection systems can provide enhanced sensitivity with reduced background

  • For HRP-conjugated primary antibodies, ensure complete blocking of endogenous peroxidases

  • Automated staining platforms can improve consistency across multiple samples

• Blocking strategy enhancement:

  • Use species-matched serum corresponding to secondary antibody host

  • Add protein blockers (BSA, casein) to reduce hydrophobic interactions

  • Consider specialized blocking reagents for endogenous biotin, peroxidase, or IgG

  • Dual blocking with both protein and detergent can reduce multiple forms of background

• Validation approaches:

  • Include known positive controls (heart, liver tissue)

  • Use mitochondrial co-markers (VDAC, COX IV) on serial sections

  • Compare staining patterns with published mitochondrial distribution patterns

  • Verify subcellular localization is consistent with mitochondrial distribution

What factors affect antibody performance in fluorescence microscopy applications?

When using NDUFB3 antibodies for immunofluorescence microscopy, several factors can significantly impact results:

• Fixation method considerations:

  • Paraformaldehyde (2-4%) preserves antigenicity while maintaining structure

  • Methanol fixation may better expose some epitopes but can distort mitochondrial morphology

  • Cold methanol (-20°C) can improve mitochondrial protein detection

  • Test multiple fixation methods with the specific NDUFB3 antibody being used

• Permeabilization optimization:

  • Mitochondrial outer membrane permeabilization requires sufficient detergent

  • Titrate detergent concentration (0.1-0.3% Triton X-100 or 0.01-0.1% saponin)

  • Longer permeabilization times may be needed for tissue sections versus cultured cells

  • Over-permeabilization can lead to epitope loss or mitochondrial disruption

• Antibody penetration strategies:

  • Extended incubation times (overnight at 4°C) improve penetration into tissue sections

  • Consider fragment antibody formats for better tissue penetration

  • Use gentle agitation during incubation to enhance antibody accessibility

  • Step-wise increasing concentrations of glycerol can improve penetration in thick sections

• Signal-to-noise optimization:

  • Autofluorescence: Pre-treat samples with sodium borohydride or Sudan Black B

  • Background: Use highly cross-adsorbed secondary antibodies

  • Photobleaching: Include anti-fade agents in mounting media

  • Signal amplification: Consider tyramide signal amplification for weak signals

• Mitochondrial network preservation:

  • Optimize fixation timing to preserve physiological mitochondrial morphology

  • Maintain sample temperature to prevent artificial fragmentation

  • Consider live-cell imaging of mitochondria before fixation for correlation

  • Use gentle washing techniques to prevent mechanical disruption

How should researchers address inconsistent NDUFB3 detection across different tissue types?

Tissue-specific variation in NDUFB3 detection requires systematic troubleshooting:

• Biological variation considerations:

  • Expression level differences: Heart tissue consistently shows strongest signals due to high mitochondrial content

  • Mitochondrial density varies across tissues (highest in heart, brain, kidney, liver)

  • Complex I assembly status may differ between tissues

  • Tissue-specific post-translational modifications may affect epitope recognition

• Protocol customization by tissue type:

  • Adjust protein loading based on expected mitochondrial content:

    • Heart: 10-15 μg total protein typically sufficient

    • Brain/Liver/Kidney: 15-20 μg recommended

    • Cultured cells: 20-30 μg often required

  • Optimize antibody concentration for each tissue type:

    • Higher dilutions (1:1000-1:8000) for heart tissue

    • Moderate dilutions (1:500-1:2000) for brain, liver, kidney

    • Lower dilutions (1:500-1:1000) for cultured cells or tissues with lower expression

• Sample preparation adaptations:

  • Tissue-specific homogenization protocols may be required (e.g., brain tissue requires gentler homogenization)

  • Adipose-rich tissues may need additional lipid removal steps

  • Fibrous tissues may require extended lysis times or mechanical disruption

  • Consider mitochondrial enrichment for tissues with lower mitochondrial content

• Quantification considerations:

  • Use tissue-specific positive controls for accurate comparison

  • Normalize to mitochondrial markers rather than total protein or cytosolic housekeeping genes

  • Consider relative quantification within tissue type rather than absolute comparison across tissues

  • Report results as percentage of control within the same tissue type to account for baseline differences

How are NDUFB3 antibodies used in the diagnosis of mitochondrial disorders?

NDUFB3 antibodies serve several diagnostic purposes in mitochondrial disease investigation:

• Diagnostic tissue analysis protocol:

  • Muscle biopsies are processed for both frozen sections and homogenate preparation

  • Western blot analysis reveals reduced NDUFB3 levels in primary defects or secondary reductions due to other Complex I deficiencies

  • Immunohistochemistry provides spatial information about NDUFB3 distribution

  • Blue Native PAGE with NDUFB3 immunodetection identifies assembly defects characterized by ~650 kDa intermediates in patients with NDUFB3 mutations

• Diagnostic algorithm implementation:

  • Initial biochemical screening shows isolated Complex I deficiency

  • NDUFB3 antibodies combined with antibodies against other Complex I subunits (NDUFA9, NDUFB8) create a "signature profile" of the defect

  • This pattern guides targeted genetic testing, potentially avoiding extensive sequencing

  • The search results indicate that recognition of characteristic facial features prompted targeted testing for the p.Trp22Arg NDUFB3 variant, circumventing invasive procedures

• Functional validation methodology:

  • Patient fibroblasts can be analyzed for NDUFB3 expression and Complex I assembly

  • This non-invasive approach complements muscle biopsy findings

  • Allows for functional validation of genetic variants of uncertain significance

  • Provides cellular material for experimental therapeutic testing

• Prognostic marker assessment:

  • Quantitative assessment of NDUFB3 and Complex I subunit levels may correlate with disease progression

  • Threshold expression levels might predict phenotypic severity

  • The search results suggest that patients with identical p.Trp22Arg mutations can have variable clinical presentations

What is the significance of NDUFB3 mutations in mitochondrial disease research?

NDUFB3 mutations provide important insights into mitochondrial biology and disease pathogenesis:

• Disease associations:

  • Isolated Complex I deficiency, the most common pediatric mitochondrial disease presentation

  • The recurrent c.64T>C, p.Trp22Arg NDUFB3 mutation has been specifically identified in multiple unrelated families

  • Initially associated with severe neurological presentations, but milder phenotypes are now recognized

• Molecular consequences:

  • Mutations cause decreased steady-state levels of multiple Complex I subunits (NDUFB8 and NDUFA9)

  • Lead to reduced fully assembled Complex I with accumulation of ~650 kDa assembly intermediates

  • Specifically affect the hydrophobic membrane arm (subcomplex Iβ) of Complex I, of which NDUFB3 is an integral component

  • This demonstrates the structural interdependence of Complex I subunits

• Genetic characteristics:

  • The c.64T>C mutation appears to have multiple independent origins rather than a single founder effect

  • Analysis of flanking STR markers identified seven distinct haplotypes associated with this mutation

  • One particularly prevalent haplotype supports a founder allele in some populations

  • This represents an interesting case of both a recurrent mutation and a founder effect

• Research implications:

  • Provides a model system for studying Complex I assembly

  • Demonstrates how a mutation in an accessory (non-catalytic) subunit can significantly impact enzyme function

  • Illustrates the importance of structural components in multi-subunit complexes

  • Highlights the complex relationship between genotype and phenotype in mitochondrial disorders

How can NDUFB3 analysis contribute to understanding phenotypic variability in mitochondrial disease?

The p.Trp22Arg NDUFB3 variant presents an intriguing model for studying factors influencing disease expression:

• Genotype-phenotype dissociation:

  • The search results reveal that patients with identical p.Trp22Arg mutations show variable clinical presentations

  • Some patients display severe neurological disease, while others have milder manifestations and good long-term prognosis

  • This suggests additional genetic or environmental modifiers

• Research methodology:

  • Quantify NDUFB3 and Complex I subunit levels across patient cohorts with identical mutations

  • Correlate protein expression with clinical severity measures

  • Perform whole genome or exome sequencing to identify potential genetic modifiers

  • Develop cellular models with controlled genetic backgrounds to isolate modifier effects

• Tissue-specific threshold effects:

  • Different tissues may have varying thresholds for mitochondrial dysfunction

  • NDUFB3 immunohistochemistry across multiple tissues from the same patient can reveal selective vulnerability

  • This approach helps explain why some organs are affected while others are spared despite an identical genetic defect

• Developmental compensation mechanisms:

  • Longitudinal studies with repeated sampling can reveal adaptive responses

  • Age-dependent alterations in NDUFB3 expression or Complex I assembly

  • Changes in mitochondrial network dynamics as compensatory mechanisms

  • This research direction may explain why some patients improve with age despite persistent genetic defects

What experimental approaches can evaluate potential therapies for NDUFB3-related disorders?

Antibody-based methods are essential for evaluating therapeutic efficacy in NDUFB3-related disorders:

• Protein replacement/stability therapy assessment:

  • Western blot quantification of NDUFB3 and other Complex I subunits before and after treatment

  • BN-PAGE analysis to determine if Complex I assembly is restored

  • Correlation with functional recovery of NADH:ubiquinone oxidoreductase activity

  • These approaches provide biochemical endpoints for clinical trials

• Gene therapy evaluation methods:

  • Immunodetection of NDUFB3 following gene delivery to confirm expression

  • Localization studies to verify proper mitochondrial targeting

  • Assembly analysis to confirm integration into functional Complex I

  • Dose-response studies to determine minimal effective expression levels

• Small molecule screening platforms:

  • High-throughput immunoassays to identify compounds that stabilize mutant NDUFB3

  • Automated imaging to quantify Complex I assembly rescue

  • Correlation of structural restoration with functional recovery

  • Mechanistic studies to determine if benefits are direct (NDUFB3 stabilization) or indirect (assembly factor modulation)

• Therapeutic biomarker development:

  • Identify NDUFB3-related proteins or metabolites that could serve as accessible biomarkers

  • Develop sensitive immunoassays for blood or urine biomarkers

  • Correlate changes in these markers with clinical improvement

  • Establish monitoring protocols for long-term follow-up of treated patients