Phospho-MAPT (S199) Recombinant Monoclonal Antibody

What is Phospho-MAPT (S199) and why is it significant in neurodegenerative research?

Phospho-MAPT (S199) refers to the microtubule-associated protein tau (MAPT) that is phosphorylated at the serine 199 residue. This specific phosphorylation event is significant in neurodegenerative research because:

• It represents one of the early phosphorylation events in the development of tau pathology

• S199 phosphorylation, particularly when combined with phosphorylation at S202 and T205 (collectively recognized by the AT8 antibody), disrupts the "paperclip" conformation of tau, potentially exposing the phosphatase-activating domain (PAD)

• The S199 site shows dramatic increases in phosphorylation at later stages of Alzheimer's disease compared to other tau residues

• It serves as a biomarker for disease progression and can be detected in various biological samples including brain tissue, cerebrospinal fluid, and plasma

• It is implicated in the formation of neurofibrillary tangles (NFTs), a hallmark pathological feature of Alzheimer's disease and other tauopathies

What are the common experimental applications for Phospho-MAPT (S199) antibodies?

Phospho-MAPT (S199) antibodies are versatile research tools with multiple applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of phosphorylated tau in solution (dilution range typically 1:2000-1:10000)

• Immunohistochemistry (IHC): For visualization of phosphorylated tau in tissue sections (typical dilution 1:50-1:200)

• Flow Cytometry (FC): For measuring phosphorylated tau in cell populations (typical dilution 1:50-1:200)

• Immunofluorescence (IF): For subcellular localization studies of phosphorylated tau (typical dilution 1:50-1:200)

• Western Blotting: For detecting phosphorylated tau protein and fragments in tissue homogenates and cellular lysates

How does the recombinant production of Phospho-MAPT (S199) antibodies compare to traditional hybridoma methods?

Recombinant production of Phospho-MAPT (S199) antibodies in expression systems like HEK293F cells offers several advantages over traditional hybridoma methods:

• Consistency: Recombinant antibodies show reduced batch-to-batch variation

• Defined Sequence: The antibody sequence is precisely known and can be engineered

• No Animal Requirements: Production doesn't require ongoing use of animals after initial sequence determination

• Scalability: Can be readily scaled up in cell culture systems

• Reduced Contaminants: Lower risk of contamination with animal pathogens or other antibodies

• Reproducibility: Enhanced experimental reproducibility due to consistent performance characteristics

The recombinant monoclonal antibodies against Phospho-MAPT (S199) are typically produced in HEK293F cells, purified by affinity chromatography, and validated for specific detection of tau phosphorylated exclusively at the S199 site .

How should researchers validate the specificity of Phospho-MAPT (S199) antibodies in their experimental system?

Validating antibody specificity is crucial for obtaining reliable results:

• Phosphatase Treatment Control: Treat duplicate samples with lambda phosphatase to remove phosphorylation and confirm loss of signal

• Peptide Competition Assay: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides spanning the S199 region to demonstrate specificity for the phosphorylated epitope

• Knockout/Knockdown Controls: Use MAPT knockout or knockdown samples as negative controls

• Multiple Antibody Validation: Compare results with other phospho-tau antibodies targeting the same site but from different sources or clones

• Immunoprecipitation-Mass Spectrometry: Confirm the identity of the immunoprecipitated protein by mass spectrometry

• Dot Blot Analysis: Test antibody against arrays of phosphorylated and non-phosphorylated tau peptides to confirm epitope specificity

What are the optimal sample preparation methods for detecting Phospho-MAPT (S199) in different experimental contexts?

Sample preparation varies by experimental application and tissue/cell type:

For Brain Tissue:

• Rapid post-mortem processing is essential to preserve phosphorylation status

• Use phosphatase inhibitors in all buffers (sodium fluoride, sodium orthovanadate, β-glycerophosphate, and sodium pyrophosphate)

• For frozen tissue: homogenize in RIPA buffer with protease and phosphatase inhibitors

• For fixed tissue: optimal fixation is 4% paraformaldehyde for 24-48 hours, followed by careful temperature-controlled paraffin embedding to preserve epitopes

For Cell Culture:

• Lyse cells directly in wells using buffer containing 20 mM Tris (pH 7.5), 0.5 mM DTT, 0.15 M NaCl, and 0.5% Triton X-100 supplemented with protease and phosphatase inhibitors

• Sonicate lysates with short pulses (4-5 seconds each) followed by centrifugation at 12,000×g for 10 minutes at 4°C

For Sarkosyl Fractionation (to separate soluble from insoluble tau):

• This method separates normal tau from aggregated pathological tau

• The ratio of Sarkosyl insoluble to Sarkosyl soluble phospho-tau can indicate the degree of pathological aggregation

What are important considerations when using Phospho-MAPT (S199) antibodies in multiplex immunoassays?

When incorporating Phospho-MAPT (S199) antibodies in multiplex assays:

• Antibody Cross-reactivity: Ensure no cross-reactivity with other phospho-epitopes or proteins in the multiplex panel

• Species Compatibility: Confirm compatibility with other antibodies in terms of species origin to avoid cross-reactivity of secondary antibodies

• Signal Optimization: Balance signal intensities across all analytes in the panel, as phospho-tau signals may be significantly weaker than other proteins

• Epitope Masking: Consider the possibility that binding of one antibody might mask nearby epitopes due to steric hindrance

• Dephosphorylation Risk: Minimize time between sample collection and assay to prevent loss of phosphorylation

• Signal Normalization: Include total tau measurement to normalize phospho-tau signals for more accurate quantification

How does phosphorylation at S199 interrelate with other tau phosphorylation sites in the pathogenesis of tauopathies?

The relationship between S199 phosphorylation and other sites involves complex temporal and functional dynamics:

• Sequential Phosphorylation: Evidence suggests that S199 phosphorylation may precede phosphorylation at other sites in the disease process, potentially acting as a priming event

• Combined Effects: When S199 is phosphorylated alongside S202 and T205 (the AT8 epitope), it causes disruption of tau's normal "paperclip" conformation, leading to exposure of the phosphatase-activating domain (PAD)

• Differential Distribution: Phosphorylation at different sites shows distinct spatial distribution patterns in the brain. While pTau-T231 is preferentially located in cytoplasm surrounding nuclei, pTau-S396 is predominantly found in nerve fibers and strongly associated with amyloid plaques

• Functional Consequences: Phosphomimetic mutations at Ser199/Ser202/Thr205 (psTau) have been shown to impair axonal transport in rat hippocampal neurons through a PAD-dependent mechanism involving protein phosphatase 1 (PP1γ)

• Biomarker Utility: In comparative studies, plasma p-tau217 typically shows stronger associations with brain amyloid-β deposition than p-tau181 and p-tau231, though phosphorylation at multiple sites including S199 increases with disease progression

Table 1: Comparison of Key Tau Phosphorylation Sites in Alzheimer's Disease

What are the methodological challenges in detecting site-specific tau phosphorylation in blood-based biomarker studies?

Blood-based detection of phosphorylated tau faces several methodological challenges:

• Low Abundance: Phosphorylated tau exists at significantly lower concentrations in blood compared to cerebrospinal fluid (CSF), requiring highly sensitive detection methods

• Matrix Effects: Plasma and serum contain various proteins and factors that can interfere with antibody binding and assay performance

• Epitope Stability: Phosphorylation at S199 may be unstable in blood samples due to phosphatase activity, necessitating rapid processing and effective phosphatase inhibitors

• Standardization Issues: Different assay platforms and antibody clones yield varying absolute values, making cross-study comparisons difficult

• Threshold Generation: Establishing clinically meaningful cutoff values requires large datasets from diverse populations

• Fragment Variability: Tau exists in multiple fragments in blood, and the phosphorylation profile may differ between fragments (e.g., 50 kDa, 38 kDa, and 25 kDa fragments show different phosphorylation patterns)

• Pre-analytical Variables: Collection tubes, processing time, freeze-thaw cycles, and storage conditions can all affect phosphorylation detection

Emerging immunoassay platforms combining immunoprecipitation with mass spectrometry (IP-MS) have shown promise for more accurate quantification of site-specific phosphorylated tau in blood samples .

How can researchers effectively model and study the functional consequences of S199 phosphorylation in experimental systems?

Several experimental approaches can be employed to study functional consequences of S199 phosphorylation:

• Phosphomimetic Mutations: Using S199E or S199D mutations to mimic constitutive phosphorylation

  • These can be introduced into tau expression constructs and studied in cell culture or animal models

  • Combined mutations at S199/S202/T205 (psTau) have been shown to disrupt tau's "paperclip" conformation and impair axonal transport

• Cell-Based Functional Assays:

  • Axonal transport assays in primary neurons to assess effects on cargo movement

  • Microtubule binding assays to determine effects on tau-microtubule interactions

  • Protein-protein interaction studies (co-immunoprecipitation) to identify altered binding partners

  • PAD exposure assays to determine conformational changes

• Kinase/Phosphatase Modulation:

  • Treatment with okadaic acid (OA) to inhibit protein phosphatases induces hyperphosphorylation of tau-S199 in wild-type mice without plaques

  • Site-specific kinase identification and inhibition studies

• Animal Models:

  • Transgenic mice expressing human tau with mutations that promote or prevent S199 phosphorylation

  • Organotypic brain slice cultures from wild-type and transgenic Alzheimer's disease mice

  • Viral vector-mediated expression of wild-type or phosphomimetic tau in specific brain regions

• Advanced Imaging:

  • FRET-based sensors to detect conformational changes associated with S199 phosphorylation

  • Live-cell imaging to track trafficking and aggregation dynamics

  • Super-resolution microscopy to visualize subcellular localization

What are common technical issues when working with Phospho-MAPT (S199) antibodies and how can they be resolved?

Researchers commonly encounter these technical challenges:

• High Background Signal

  • Cause: Non-specific binding, excessive antibody concentration, inadequate blocking

  • Solution: Optimize antibody dilution (start with 1:200 for IHC/IF), extend blocking time, use alternative blockers (5% BSA or 5% normal serum from secondary antibody species)

• Weak or Absent Signal

  • Cause: Epitope masking, dephosphorylation during processing, insufficient antigen retrieval

  • Solution: Ensure phosphatase inhibitors are fresh and used at appropriate concentrations; for IHC, try heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Non-specific Bands in Western Blot

  • Cause: Cross-reactivity, protein degradation, non-specific binding

  • Solution: Include appropriate controls (phosphatase-treated samples), optimize washing steps, try alternative blocking agents

• Variable Results Between Experiments

  • Cause: Batch-to-batch antibody variation, inconsistent sample handling

  • Solution: Use the same antibody lot when possible, standardize all protocols, include positive control samples in every experiment

• Poor Reproducibility in Quantitative Assays

  • Cause: Phosphorylation instability, assay variability

  • Solution: Establish tight time controls for sample processing, include internal calibrators, perform technical replicates

How should researchers interpret conflicting data when comparing different phospho-tau detection methods?

When faced with conflicting data from different detection methods:

• Understand Methodological Differences:

  • Different antibodies may have varying specificities and affinities even when targeting the same epitope

  • Detection platforms have different sensitivity thresholds and dynamic ranges

  • Sample preparation can affect epitope availability

• Consider Technical Validation:

  • Compare results using multiple antibody clones against the same phospho-epitope

  • Assess antibody specificity using phosphatase-treated controls and peptide competition assays

  • Validate findings using orthogonal methods (e.g., mass spectrometry)

• Evaluate Sample-Specific Factors:

  • Tau fragments may vary between sample types, affecting epitope presentation

  • Matrix effects can influence antibody binding in complex biological samples

  • Post-translational modifications beyond phosphorylation may mask epitopes

• Establish Context-Specific Benchmarks:

  • In plasma biomarker studies, IP-MS-based methods for p-tau217 have shown superior performance compared to immunoassay-based approaches

  • Different detection methods may be optimal for different applications

• Report Comprehensive Methodological Details:

  • Include complete information about antibody clones, dilutions, incubation conditions

  • Specify exact sample processing steps, including timing and temperature

  • Report all controls and validation steps performed

What are the most reliable methods for quantifying relative changes in S199 phosphorylation across experimental conditions?

For reliable quantification of relative changes:

• Western Blotting with Dual Detection:

  • Probe for phospho-S199 and total tau simultaneously (using differently labeled secondary antibodies)

  • Calculate the ratio of phospho-S199 to total tau for each sample

  • Include a concentration gradient of a standard sample to ensure linearity of detection

• ELISA-Based Approaches:

  • Sandwich ELISA with capture antibody against total tau and detection antibody against phospho-S199

  • Include a standard curve using recombinant phospho-tau

  • Normalize to total tau measured in parallel assays

• Quantitative Immunofluorescence:

  • Co-stain with antibodies against phospho-S199 and total tau

  • Use automated image analysis to calculate mean intensity ratios

  • Include internal control regions to normalize between sections/samples

• Mass Spectrometry-Based Methods:

  • Targeted MS approaches (multiple reaction monitoring) for absolute quantification

  • Immunoprecipitation followed by MS for enhanced sensitivity

  • Isotope-labeled internal standards for accurate quantification

• Flow Cytometry:

  • Dual staining for phospho-S199 and total tau

  • Calculate mean fluorescence intensity ratios

  • Include calibration beads to standardize between experiments

How can Phospho-MAPT (S199) antibodies be utilized in developing therapeutic approaches for tauopathies?

Phospho-MAPT (S199) antibodies play crucial roles in therapeutic development:

• Target Validation:

  • Confirming the presence and abundance of phospho-S199 tau in patient samples

  • Correlating phospho-S199 levels with disease severity and progression

• Therapeutic Antibody Development:

  • Serving as templates for therapeutic antibody engineering

  • Competing with therapeutic candidates in binding assays to confirm epitope targeting

• Vaccine Development Assessment:

  • Evaluating antibody responses to tau-targeted vaccines

  • The virus-like particle (VLP)-based vaccines targeting phosphorylated tau epitopes (like Qβ-AT8, which includes the S199 site) have shown promise in reducing tau pathology

• Drug Screening:

  • Quantifying changes in phospho-S199 levels in response to kinase or phosphatase modulators

  • High-throughput screening assays to identify compounds that reduce S199 phosphorylation

• Biomarker for Clinical Trials:

  • Monitoring treatment effects on phospho-tau levels in biological fluids

  • Participant stratification based on baseline phospho-tau profiles

• Target Engagement Studies:

  • Confirming that therapeutic interventions effectively engage with phosphorylated tau

  • Determining dose-response relationships for investigational treatments

What is the significance of S199 phosphorylation in relation to tau's role in axonal transport disruption?

S199 phosphorylation has specific implications for axonal transport:

• PAD Exposure Mechanism:

  • Phosphorylation at S199, especially in combination with S202 and T205 (psTau), disrupts tau's "paperclip" conformation

  • This disruption exposes the phosphatase-activating domain (PAD) at the N-terminus

  • Exposed PAD enhances interactions with protein phosphatase 1 gamma (PP1γ)

• PP1γ Activation and Consequences:

  • Phosphomimetic mutations at S199/S202/T205 (psTau) increase active PP1γ levels in mammalian cells

  • This activation occurs through direct interaction between exposed PAD and PP1γ

  • The activation of PP1γ triggers signaling cascades that inhibit anterograde fast axonal transport

• Experimental Evidence:

  • Expression of psTau causes significant impairment of axonal transport in primary rat hippocampal neurons

  • Deletion of PAD in psTau significantly reduces interaction with PP1γ and rescues axonal transport impairment

  • Similar effects have been observed in squid axoplasm models with phosphomimetic mutations

• Disease Relevance:

  • Axonal transport disruption is an early event in Alzheimer's disease and other tauopathies

  • The PAD-dependent mechanism may represent a potential therapeutic target

  • Compounds that prevent PAD exposure or block PAD-PP1γ interaction might protect against transport deficits

How are plasma phospho-tau measurements, including S199, being developed as biomarkers for Alzheimer's disease diagnosis and monitoring?

Plasma phospho-tau biomarkers are rapidly evolving:

• Diagnostic Applications:

  • Plasma p-tau forms correlate with cognitive capacity assessed with various instruments including MMSE, MoCA, and CDR-SOB

  • Baseline plasma p-tau concentrations predict future cognitive decline and progression to MCI and dementia

  • Performance sometimes parallels that of CSF p-tau measurements

• Monitoring Disease Progression:

  • Increased levels of plasma p-tau associate with more rapid decline in cognition, cortical thickness, hippocampal atrophy, and glucose metabolism

  • Longitudinal measurements can track disease progression or treatment effects

• Technical Advancements:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS) technology has shown superior performance for p-tau217 compared to traditional immunoassay methods

  • Single molecule array (Simoa) technology allows detection of extremely low concentrations of phospho-tau in plasma

• Differential Diagnosis:

  • In participants carrying MAPT mutations that cause non-AD tauopathies, blood-based p-tau181 levels remained normal as in healthy controls

  • This suggests potential specificity for Alzheimer's-type tau pathology versus other tauopathies

• Special Populations:

  • In Down syndrome, which has high rates of Alzheimer's pathology, plasma p-tau181 and p-tau217 can discriminate asymptomatic individuals from those with prodromal and dementia stages

  • Plasma p-tau181 in Down syndrome correlates with atrophy and hypometabolism in temporoparietal regions

Table 2: Comparison of Different Plasma Phospho-Tau Biomarkers in Alzheimer's Disease

How should researchers interpret changes in Phospho-MAPT (S199) levels in different experimental models of neurodegeneration?

Interpretation of phospho-S199 changes requires contextual understanding:

• Model-Specific Considerations:

  • Transgenic Mouse Models: Different tau transgenic lines show varying phosphorylation profiles and progression rates

  • Cell Culture Models: Neuronal versus non-neuronal cells may show different phosphorylation regulation

  • Acute vs. Chronic Models: Acute treatments (like okadaic acid) may induce different phosphorylation patterns than chronic disease models

• Temporal Dynamics:

  • Early increases may represent physiological stress responses

  • Persistent elevation suggests pathological processes

  • The relationship between S199 phosphorylation and other sites may shift over disease course

• Regional Variations:

  • Hippocampal and cortical regions may show different phosphorylation patterns

  • Cellular distribution (soma vs. neurites) provides important functional context

• Relationship to Other Pathologies:

  • Correlation with amyloid pathology in AD models

  • Presence or absence of insoluble tau aggregates (Sarkosyl-insoluble fraction)

  • Evidence of neurodegeneration (synaptic loss, neuronal death)

• Molecular Context:

  • PAD exposure status as a functional readout of S199 phosphorylation

  • Effects on protein-protein interactions, particularly with phosphatases like PP1γ

  • Consequences for microtubule binding and axonal transport

What are the current limitations in our understanding of the relationship between S199 phosphorylation and tau pathology?

Several knowledge gaps persist in our understanding:

How does the interaction between Phospho-MAPT (S199) and protein phosphatase 1 gamma (PP1γ) contribute to tau-mediated neurodegeneration?

The phospho-S199-PP1γ interaction reveals a specific mechanism of pathology:

• Molecular Mechanism:

  • Phosphorylation at S199 (especially in combination with S202/T205) disrupts tau's "paperclip" conformation

  • This conformational change exposes the N-terminal phosphatase-activating domain (PAD)

  • Exposed PAD enhances interaction with PP1γ, as demonstrated by co-immunoprecipitation experiments

  • This interaction increases levels of active PP1γ in cells

• Functional Consequences:

  • Activated PP1γ triggers signaling cascades that impair anterograde fast axonal transport

  • This leads to disruption of cargo delivery to synapses and axon terminals

  • Expression of phosphomimetic tau at S199/S202/T205 (psTau) impairs axonal transport in primary rat hippocampal neurons

  • Deletion of PAD rescues this transport impairment, confirming the PAD-dependent mechanism

• Amplification Cycle:

  • Transport deficits may further compromise neuronal health

  • Compromised neurons may exhibit altered kinase/phosphatase balance

  • This could lead to additional tau phosphorylation, creating a pathological cycle

• Therapeutic Implications:

  • Targeting the tau-PP1γ interaction or downstream signaling represents a potential intervention strategy

  • Compounds that prevent PAD exposure or block its interaction with PP1γ might protect against transport deficits

  • Small molecule inhibitors of this protein-protein interaction could be therapeutic candidates

Figure 1: Model of S199 Phosphorylation-Induced PAD Exposure and PP1γ Activation

The figure would illustrate the proposed mechanism where tau phosphorylation at S199 (along with S202/T205) disrupts the paperclip conformation, exposing PAD, which then interacts with and activates PP1γ, leading to axonal transport impairment and subsequent neurodegeneration.