THPO Antibody

What is THPO and why is it an important research target?

Thrombopoietin (THPO, also known as TPO, MGDF, or MPL ligand) is a lineage-specific cytokine primarily responsible for the generation of platelets through its action on hematopoietic cells committed to the megakaryocyte lineage. THPO is constitutively produced by the liver and has a predicted molecular weight of approximately 38 kDa . When THPO binds to its receptor, MPL (CD110), it initiates signaling cascades via JAK/STAT, MAPK, and PI3K/AKT pathways that lead to the proliferation and differentiation of megakaryocytes and ultimately to platelet production (thrombopoiesis) . Dysregulation of THPO has been linked to various hematological disorders, including thrombocytopenia and myeloproliferative neoplasms, making it a significant target for research in hematology and oncology .

What types of THPO antibodies are available for research?

Research-grade THPO antibodies are available in several formats:

Both types have specific advantages: monoclonal antibodies offer high specificity and reproducibility, while polyclonal antibodies can provide broader epitope recognition .

What are the common applications for THPO antibodies in research?

THPO antibodies are used in multiple experimental applications:

• Western blot (typically at dilutions of 1:500-1:5000)

• ELISA (typically at dilutions of 1:2000-1:10000)

• Flow cytometry (typically at dilutions of 1:200-1:400)

• CyTOF (mass cytometry)

These applications enable researchers to detect and quantify THPO expression in various tissues and cell types, particularly in hematopoietic stem cells and megakaryocyte lineages.

How should THPO antibodies be stored and handled to maintain optimal activity?

For optimal preservation of THPO antibody activity:

• Store at 4°C for short-term use (days to weeks)

• For long-term storage, aliquot and store at -20°C to avoid repeated freeze-thaw cycles

• Most commercial THPO antibodies are supplied in buffers containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% glycerol in PBS, pH 7.4)

• When thawing frozen aliquots, bring to room temperature gradually and mix gently to avoid protein denaturation

• Always centrifuge briefly before opening vials to collect liquid that may have gathered on the cap or sides

What are the optimal protocols for detecting THPO using antibodies in Western blot?

Recommended Western blot protocol for THPO detection:

• Sample preparation: Use whole cell lysates from THPO-expressing tissues (e.g., liver) or cell lines (e.g., 293T cells)

• Gel electrophoresis: Load 20-50 μg protein per lane on 10-12% SDS-PAGE gels

• Transfer: Standard wet transfer to PVDF membrane at 100V for 1 hour

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute THPO antibody at 1:1000-1:5000 in blocking buffer; incubate overnight at 4°C

• Washing: 3 × 10 minutes with TBST

• Secondary antibody: Anti-species IgG-HRP at 1:10000 dilution for 1 hour at room temperature

• Detection: Enhanced chemiluminescence (ECL)

Expected results: THPO typically appears as a band of approximately 35-38 kDa . Additional bands may represent glycosylated forms or processing intermediates.

How can I optimize ELISA protocols for THPO detection?

For THPO ELISA optimization:

• Coating: Use capture antibody at 1-2 μg/ml in carbonate buffer (pH 9.6); incubate overnight at 4°C

• Blocking: 2-3% BSA in PBS for 1-2 hours at room temperature

• Sample preparation: Dilute serum samples 1:2 to 1:10 in sample diluent

• Detection antibody: Use biotinylated anti-THPO at 1:2000-1:10000 dilution

• Signal development: Streptavidin-HRP followed by TMB substrate

• Sensitivity enhancement: Consider amplification systems for low abundance samples

• Validation: Include recombinant THPO standards (5-500 pg/ml) for quantification

How can THPO antibodies be used to study hematopoietic stem cell populations?

THPO antibodies are valuable tools for investigating hematopoietic stem cell (HSC) biology:

• Surface marker analysis: Use flow cytometry with THPO receptor (MPL) antibodies to identify HSC populations with megakaryocytic potential

• Lineage tracing: Combined with CD41, CD150, and CD48 markers, THPO/MPL staining can distinguish between HSC subpopulations with different megakaryopoietic potential

• Cell sorting: THPO receptor expression can be used to isolate HSCs with high thrombopoietic capacity

• Functional assays: THPO antibodies can neutralize THPO activity in culture systems to assess dependence of specific HSC populations on THPO signaling

Research has shown that CD150+ HSPCs and LT-HSCs are highly dependent on THPO signaling, while CD150- HSPCs show reduced dependence, correlating with their lower MPL expression levels .

What role do anti-THPO autoantibodies play in thrombocytopenia, and how can they be studied?

Anti-THPO autoantibodies have been identified in several clinical conditions:

• Detection methods:

  • Custom ELISA using plates coated with recombinant THPO

  • ELISA with GSH-treated microtiter plates for capturing THPO

  • Neutralization assays using BaF3 cells expressing the THPO receptor

• Clinical significance:

  • Found in patients with amegakaryocytic thrombocytopenic purpura, idiopathic thrombocytopenia purpura (ITP), and systemic lupus erythematosus (SLE)

  • Present in approximately 16% of patients with type 2 diabetes mellitus (T2DM)

  • Associated with significantly lower platelet counts (multivariate linear regression analysis showed β = −0.23; p < 0.05)

• Mechanistic studies:

  • Anti-THPO autoantibodies may block normal THPO function, resulting in decreased platelet production

  • May be induced by chronic inflammation, with T-cells in T2DM patients showing increased production of pro-inflammatory cytokines

How can THPO antibodies be used in developability assessment of therapeutic antibodies?

When developing therapeutic antibodies (including anti-THPO antibodies), multiple biophysical properties must be assessed:

• Critical antibody properties to evaluate:

  • Colloidal properties (aggregation, self-interaction, hydrophobicity, viscosity)

  • Fragmentation/clipping susceptibility

  • Post-translational modifications (PTMs)

  • Charge (pI)

  • Thermal stability

• High-throughput assessment protocol:

  • Express antibodies transiently in CHO-Expi cells

  • Purify via automated protein A chromatography (100 μgs – ~1 mg scales)

  • Characterize using surface plasmon resonance (SPR) and functional assays

  • Analyze biophysical properties predictive of downstream behavior

• Specific THPO antibody considerations:

  • Evaluate binding to both glycosylated and non-glycosylated forms of THPO

  • Assess cross-reactivity with THPO from different species (human, mouse, rat)

  • Test for neutralizing capacity in functional assays

What are common issues with THPO antibody detection, and how can they be resolved?

Common problems and solutions in THPO antibody experiments:

How can I distinguish between anti-THPO autoantibodies and normal variation in platelet counts?

To accurately identify and study anti-THPO autoantibodies:

• Establish a reliable detection method:

  • Develop a quantitative ELISA specifically for anti-THPO autoantibodies

  • Include positive controls from known positive samples

  • Establish a clear cutoff value based on healthy control populations

• Account for confounding factors:

  • Age (negatively correlated with platelet count)

  • HbA1c levels (positively correlated with platelet count)

  • White blood cell count (positively correlated with platelet count)

  • Mean platelet volume (MPV, negatively correlated with platelet count)

• Statistical approach:

  • Perform multivariate linear regression analyses to determine independent association between anti-THPO antibodies and platelet counts

  • Research has shown that anti-THPO antibody presence remains significantly associated with decreased platelet counts (β = −0.23; p < 0.05) even after adjusting for other variables

What considerations should be made when using THPO antibodies across different species?

Cross-species reactivity considerations:

• Species homology:

  • Human and mouse THPO share approximately 70% amino acid sequence identity

  • Epitope conservation varies across regions of the protein

• Validated reactivity:

  • Some antibodies are species-specific (human-only)

  • Others show cross-reactivity (e.g., PACO32440 reacts with human, mouse, and rat THPO)

• Experimental validation:

  • Always perform positive controls with recombinant THPO from the species of interest

  • Use tissues known to express high levels of THPO (liver) from the target species

  • Consider using knockout/knockdown controls when available

• Application sensitivity:

  • Western blot often shows more cross-reactivity than immunohistochemistry

  • For flow cytometry, species-specific antibodies are strongly recommended

How are THPO antibodies being used to study the interaction between Th/To antibodies and systemic sclerosis?

Recent research has revealed significant connections between anti-Th/To antibodies and systemic sclerosis (SSc):

• Clinical significance:

  • Anti-Th/To positive SSc patients show a distinct clinical profile with higher rates of pulmonary hypertension (PH) (38% vs. 15% in controls, p<0.0001)

  • These patients have higher rates of WHO Group 1 pulmonary arterial hypertension (PAH) (23% vs. 9% in controls, p<0.0001)

  • Anti-Th/To antibody presence is associated with a 3.3-fold increased risk of developing PH at 10 years of follow-up

• Research methodology:

  • Case-control studies with long-term follow-up (median 6.1 years)

  • Anti-Th/To antibody detection methods must be standardized, as these were historically accessible only to a few research centers

• Potential mechanistic connections:

  • Both Th/To and THPO antibodies affect hematopoietic pathways

  • Standardized THPO antibody-based assays could help investigate potential overlap or interaction between these autoantibody systems

How can inverse folding models improve THPO antibody design and functionality?

Advanced computational approaches are transforming antibody design:

• Inverse folding technology:

  • Models like AntiFold can predict antibody sequences based on structural constraints

  • Can achieve 60% amino acid recovery for CDRH3 loops

  • Helps maintain structural integrity while optimizing properties

• Application to THPO antibodies:

  • Structure-based design can optimize THPO antibody binding while maintaining backbone structure

  • Helps identify variants with improved binding affinity without disrupting other properties

  • Enables rational optimization of complementarity-determining regions (CDRs)

• Prediction capabilities:

  • Inverse folding models can predict binding affinity, stability, and developability

  • Log-likelihood calculations from these models correlate with experimental binding measurements

  • Can identify high-fitness, structurally constrained regions of the mutational landscape

• Integration with experimental data:

  • Combine computational predictions with experimental validation

  • Use structure-activity relationship data to refine models

  • Implement iterative design-build-test cycles for optimization

What methodological advances are improving the detection and characterization of anti-THPO antibodies in clinical samples?

Emerging methodologies for anti-THPO antibody research:

• Advanced detection techniques:

  • Multiplex bead-based assays for simultaneous detection of multiple autoantibodies

  • Single B-cell sequencing to identify anti-THPO antibody-producing clones

  • Quantitative mass spectrometry for antibody characterization

• Functional assessments:

  • Cell-based neutralization assays using BaF3 cells expressing THPO receptor

  • Megakaryocyte differentiation assays to evaluate functional impact

  • In vitro thrombopoiesis models to assess effects on platelet production

• Integration with clinical data:

  • Machine learning approaches to identify clinical patterns associated with anti-THPO antibodies

  • Longitudinal studies to monitor antibody levels and correlate with disease progression

  • Multi-center validation studies to establish standardized reference ranges

• Standardization efforts:

  • Development of international reference standards for anti-THPO antibodies

  • Harmonization of assay protocols across laboratories

  • Establishment of clinically relevant cutoff values