CNTFR Antibody

What is CNTFR and what are its key structural and functional characteristics?

CNTFR (Ciliary Neurotrophic Factor Receptor) is a hematopoietin/interferon-class receptor belonging to the cytokine superfamily of receptors. It functions as the specific alpha subunit of a heterotrimeric receptor complex that also includes LIFR and gp130 . Key characteristics include:

• Structure: Contains an immunoglobulin-like C2-type domain and a fibronectin type-III domain

• Membrane Attachment: Attached to the cell membrane via a glycosyl-phosphatidylinositol (GPI) linkage

• Molecular Weight:

  • Calculated: 41 kDa

  • Observed: 45-50 kDa (due to post-translational modifications)

• Signaling Mechanism: CNTF must first bind to the alpha component (CNTFR), which then permits recruitment of gp130 and LIFR beta to form the tripartite receptor complex

• Biological Functions: Signal transduction through this complex stimulates gene expression, cell survival, and differentiation in various neuronal cell types

Different applications require specific antibody dilutions for optimal results:

It is recommended that each laboratory determine optimal dilutions for their specific experimental system, as results may vary depending on sample type and preparation .

How can CNTFR antibodies be effectively used in neutralization assays?

CNTFR antibodies are valuable tools for neutralization assays to study receptor-ligand interactions. The methodology involves:

• Experimental Setup:

  • Use recombinant CNTFR to enhance CNTF-dependent cell proliferation (e.g., in TF-1 human erythroleukemic cell line)

  • Add increasing concentrations of anti-CNTFR antibody to neutralize this enhancement

• Protocol Details:

  • For human systems: Use 1 μg/mL recombinant human CNTFR alpha and 20 ng/mL recombinant human CNTF

  • For rat systems: Use 0.25 μg/mL recombinant rat CNTFR alpha and 10 ng/mL recombinant rat CNTF

  • Add anti-CNTFR antibody at increasing concentrations

  • Measure cell proliferation to determine neutralization effectiveness

• Expected Results:

  • Human system: ND50 typically 10-20 μg/mL with AF-303-NA antibody

  • Rat system: ND50 typically 2-6 μg/mL with AF-559-NA antibody

  • Results should show dose-dependent neutralization of CNTF-induced proliferation

• Controls:

  • Include cells treated with CNTF alone

  • Include cells treated with CNTF and CNTFR without antibody

  • Include appropriate isotype control antibodies

This approach allows researchers to quantitatively assess the role of CNTFR in CNTF-mediated signaling pathways and evaluate antibody effectiveness.

What are the validated methods for detecting CNTFR expression in tissue samples?

Detection of CNTFR in tissues requires optimized protocols for accurate results:

• Immunohistochemistry (IHC):

  • Sample Preparation: Use immersion-fixed paraffin-embedded sections

  • Epitope Retrieval: Heat-induced epitope retrieval with basic retrieval reagent (e.g., VisUCyte Antigen Retrieval Reagent-Basic)

  • Antibody Incubation: 5 μg/mL MAB303 for 1 hour at room temperature

  • Detection System: Anti-Mouse IgG HRP Polymer Antibody

  • Visualization: DAB (brown) with hematoxylin counterstain (blue)

  • Expected Results: In skeletal muscle, specific staining localized to sarcolemma and sarcoplasm

• Immunofluorescence/Immunocytochemistry:

  • Useful for co-localization studies with other markers

  • Example application: Detection of CNTFR in tyrosine hydroxylase (TH)+ processes extending toward ventricular walls in neuronal tissue

  • Can be combined with markers of neuronal activation (c-Fos) to study functional responses

• In Situ Hybridization:

  • For detecting CNTFR mRNA expression

  • Has been used to show expression gradients in structures like mouse locus coeruleus

  • Provides spatial resolution of expression patterns

• Western Blotting:

  • Validated tissues: mouse brain, human brain, mouse cerebellum, rat brain tissue

  • Validated cell lines: SH-SY5Y cells

  • Expected molecular weight: 45-50 kDa

Each method requires proper validation with appropriate positive and negative controls to ensure specificity of CNTFR detection.

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

Proper storage and handling of CNTFR antibodies is crucial for maintaining their activity:

Additional handling considerations:

• Avoid repeated freeze-thaw cycles

• Centrifuge vial briefly before opening to ensure recovery of all material

• When preparing working dilutions, use fresh buffers free of contaminants

• For critical applications, pre-clear antibody solutions by centrifugation to remove potential aggregates

How do CNTFR antibodies help elucidate complex signaling mechanisms between CNTFR and its binding partners?

CNTFR antibodies serve as critical tools for investigating the intricate signaling mechanisms involving CNTFR and its multiple binding partners:

• Co-immunoprecipitation Studies:

  • Anti-CNTFR antibodies can be used to immunoprecipitate CNTFR complexes

  • This approach has revealed that CNTFR forms heterodimers with CLC (cardiotrophin-like cytokine) that can be co-precipitated

  • The analysis showed that CLC and CNTFR specifically associate to form a secreted heterodimer with biological activity

• Complex Formation Analysis:

  • HPLC gel filtration combined with western blotting using anti-CNTFR antibodies has determined that:

    • The CLC-sCNTFR complex elutes with an apparent molecular weight of 120-150 kDa

    • Only about 30% of soluble CNTFR is engaged in composite cytokine formation

    • The complex likely consists of two molecules each of CLC (22-25 kDa) and sCNTFR (50 kDa)

• Functional Signaling Studies:

  • Anti-CNTFR antibodies can neutralize receptor function to assess downstream effects

  • Studies have demonstrated that the CLC-sCNTFR complex activates the LIF receptor (gp130 and LIFR) and the tripartite CNTF receptor (gp130, LIFR, and CNTFR)

  • The specific activities were measured at 2 × 10^5 and 5 × 10^7 U/mg of protein, respectively

• Receptor Binding Site Mapping:

  • Mutational studies combined with antibody binding assays have identified that:

    • Site I binding is critical for CLC-CNTFR association to generate a secreted composite cytokine

    • Mutations in positions 170 and 174 (C-terminal part of helix D) and position 60 (AB loop) completely prevent CLC secretion when co-expressed with sCNTFR

These approaches collectively provide comprehensive insights into the molecular mechanisms governing CNTFR-mediated signaling in various biological contexts.

What strategies can be employed to engineer and optimize CNTFR-targeting molecules for therapeutic applications?

Engineering CNTFR-targeting molecules involves sophisticated approaches to modulate receptor activity:

• Yeast Surface Display for Affinity Maturation:

  • This combinatorial protein engineering method has been used to generate CLCF1 variants with altered CNTFR binding properties

  • The process involves displaying CLCF1 on yeast cell surfaces as fusions to Aga2p mating protein

  • Flow cytometry quantification of binding to CNTFR-Fc fusion proteins allows for selection of high-affinity variants

• Directed Evolution Approach:

  • First-generation library creation: Random mutations introduced via error-prone PCR (~1 × 10^8 yeast transformants)

  • Selection process: Iterative rounds of fluorescence-activated cell sorting (FACS) to isolate variants with top 0.5-1% binding signal

  • Refinement: Increasing stringency by decreasing CNTFR-Fc concentration in subsequent sorts

  • DNA shuffling: Second-generation libraries created by shuffling enriched variants using staggered extension process (StEP)

• Key Mutations for Enhanced Binding:

  • First-generation consensus mutation: Q96R

  • Additional beneficial mutations: L86F, H148R (increasing binding affinity ~2-fold)

  • Second-generation consensus mutations: W169L and K180R (located in purported CNTFR binding region)

• Engineered Functional Variants:

  • Superagonists: Variants with increased CNTFR affinity that enhance tripartite receptor complex formation and signaling

  • Antagonists: Variants that bind CNTFR with high affinity but do not engage coreceptors (gp130 or LIFR), functioning as receptor blockers

  • Applications include potential treatments for nonsmall cell lung cancer (NSCLC) and promotion of axon regeneration

These engineering strategies provide a foundation for developing novel therapeutics targeting the CLCF1-CNTFR signaling axis with precisely controlled functional outcomes.

How can CNTFR antibodies be utilized to investigate neuroinflammatory processes and microglial activation?

CNTFR antibodies serve as valuable tools for studying neuroinflammatory processes:

• Microglia Activation Studies:

  • CNTF has been shown to activate microglia and dendritic-like microglia in ways similar to IL-6

  • Anti-CNTFR antibodies can help distinguish direct effects of CNTF on microglia from indirect effects

  • Research has demonstrated that CNTF-treated rat microglia secrete soluble factors that increase motor neuron survival

• Neuroinflammation Assessment Techniques:

  • Immunohistochemical Analysis:

    • CNTFR antibodies can identify receptor expression on different CNS cell types

    • Co-staining with microglial markers (Iba1, CD11b) helps determine which cell populations respond to CNTF

    • Changes in microglial morphology (from ramified to ameboid) can be quantified in response to CNTF treatment

• Mechanistic Investigation:

  • CNTFR antibodies can block receptor function to determine:

    • If microglial activation is directly mediated by CNTFR

    • Which downstream signaling pathways are activated

    • How CNTFR signaling differs from other related cytokine receptors

• Ex Vivo Applications:

  • Studies using LC explants acutely exposed to recombinant CNTF have demonstrated:

    • Increased TH (tyrosine hydroxylase) phosphorylation

    • Enhanced Erk1 phosphorylation

    • These effects can be blocked using CNTFR antibodies to confirm specificity

• Functional Outcomes:

  • CNTFR antibodies can help determine how CNTF-induced microglial activation affects:

    • Neurotrophic factor production

    • Phagocytic activity

    • Inflammatory cytokine release

    • Neuronal survival and function

This multifaceted approach allows researchers to dissect the complex role of CNTFR in neuroinflammatory processes and its potential as a therapeutic target for neurodegenerative conditions.

What are common technical challenges when using CNTFR antibodies, and how can researchers address them?

Researchers may encounter several challenges when working with CNTFR antibodies:

• Variable Signal Intensity in Western Blots:

  • Challenge: Inconsistent or weak CNTFR detection

  • Solutions:

    • Optimize protein extraction methods for membrane-associated GPI-linked proteins

    • Try different lysis buffers containing appropriate detergents

    • Adjust antibody concentration (recommended range: 1:1000-1:6000 for WB)

    • Increase protein loading amount for low-expressing samples

    • Try different blocking agents to reduce background

• Cross-Reactivity Issues:

  • Challenge: Nonspecific binding or unexpected bands

  • Solutions:

    • Verify antibody specificity with positive and negative control samples

    • Use tissues known to express CNTFR (brain, skeletal muscle) as positive controls

    • Include CNTFR knockout or knockdown samples as negative controls

    • Perform peptide competition assays to confirm specificity

    • Consider the ~50% cross-reactivity with recombinant human CNTF sR alpha when using certain antibodies

• Immunohistochemistry Optimization:

  • Challenge: Poor staining or high background

  • Solutions:

    • Test different antigen retrieval methods:

      • TE buffer pH 9.0 (recommended)

      • Alternative: citrate buffer pH 6.0

    • Optimize antibody concentration (1:50-1:500 range for IHC)

    • Extend incubation time at lower antibody concentrations

    • Use appropriate detection systems (e.g., Anti-Mouse IgG HRP Polymer)

• Functional Assay Variability:

  • Challenge: Inconsistent neutralization results

  • Solutions:

    • Ensure recombinant proteins are active and properly folded

    • Titrate both recombinant CNTFR and CNTF concentrations

    • Verify endotoxin levels (<0.10 EU per 1 μg) to avoid non-specific effects

    • Use freshly prepared reagents and consistent cell passage numbers

Each laboratory should conduct pilot studies to optimize conditions for their specific experimental system, as sample types and preparation methods can significantly impact results.

How can researchers validate the specificity and functional activity of CNTFR antibodies?

Comprehensive validation ensures reliable CNTFR antibody performance:

• Specificity Validation:

  Validation Method	Procedure	Expected Outcome
  Western Blot	Run samples from known CNTFR-expressing tissues (brain, muscle)	Single band at 45-50 kDa
  Peptide Competition	Pre-incubate antibody with immunizing peptide	Significant reduction in signal
  Genetic Models	Test samples from CNTFR knockout/knockdown models	Absence or reduction of signal
  Immunoprecipitation-Mass Spectrometry	IP followed by MS identification	Confirmation of CNTFR peptides

• Functional Activity Assessment:

  Assay Type	Methodology	Validation Criteria
  Neutralization	TF-1 cell proliferation with CNTF+CNTFR, +/- antibody	Dose-dependent inhibition with ND50 of 10-20 μg/mL (human) or 2-6 μg/mL (rat)
  Immunoprecipitation	IP of CNTFR complexes from cells	Co-precipitation of known binding partners (e.g., CLC)
  Receptor Signaling	Treatment of cells with CNTF +/- antibody pre-treatment	Inhibition of phosphorylation of downstream targets (e.g., STAT3, Erk1/2)

• Cross-Platform Validation:

  • Verify CNTFR detection across multiple techniques (WB, IHC, IF, ELISA)

  • Compare results obtained with different CNTFR antibody clones

  • Correlate protein detection with mRNA expression data

• Reproducibility Testing:

  • Analyze lot-to-lot variation by testing multiple antibody batches

  • Assess inter-laboratory reproducibility through collaborative testing

  • Document consistent results across different experimental conditions

Thorough validation not only confirms antibody reliability but also establishes optimal parameters for subsequent experiments, ensuring scientific rigor and reproducibility.

What approaches can address discrepancies in experimental results when using different CNTFR antibody clones?

When faced with discrepancies between different CNTFR antibody clones, researchers should implement systematic troubleshooting:

• Epitope Mapping and Comparison:

  • Determine the epitopes recognized by each antibody clone

  • Different antibodies may target distinct domains of CNTFR:

    • Some target the immunoglobulin-like C2-type domain

    • Others target the fibronectin type-III domain

  • Epitope accessibility may vary depending on sample preparation and experimental conditions

• Systematic Parallel Testing:

  • Run side-by-side experiments with multiple antibody clones

  • Use standardized protocols and identical samples

  • Document differences in:

    • Signal intensity

    • Specificity (number of bands/background)

    • Optimal working concentrations

    • Buffer compatibility

• Post-Translational Modification Considerations:

  • CNTFR undergoes various post-translational modifications

  • The calculated molecular weight (41 kDa) differs from observed (45-50 kDa)

  • Different antibodies may have variable sensitivity to:

    • Glycosylation states

    • Phosphorylation

    • GPI-anchor modifications

    • Proteolytic processing

• Reconciliation Strategies:

  • Orthogonal Validation: Confirm results using non-antibody methods (e.g., mass spectrometry)

  • Knockout/Knockdown Controls: Test antibodies on samples with genetically reduced CNTFR expression

  • Functional Correlation: Correlate antibody binding with functional outcomes

  • Complementary Approaches: Use combinatorial detection with multiple antibodies targeting different epitopes

• Reporting and Documentation:

  • Thoroughly document all antibody details in publications:

    • Catalog number

    • Clone ID

    • Lot number

    • Epitope information

    • Validation methods

  • This enables better reproducibility and helps the research community interpret seemingly conflicting results

By implementing these approaches, researchers can better understand the source of discrepancies and select the most appropriate antibody for their specific application, enhancing data reliability and experimental reproducibility.

How are CNTFR antibodies being utilized in current research on neurodegeneration and neuroprotection?

CNTFR antibodies are enabling significant advances in understanding neurodegeneration and developing neuroprotective strategies:

• Mechanistic Studies of Neuroprotection:

  • CNTFR antibodies help elucidate how CNTF provides neuroprotection

  • Research has shown that CNTF-treated rat microglia secrete soluble factors that increase motor neuron survival

  • Antibody-based blocking studies reveal which specific pathways mediate these effects

• Receptor Complex Formation and Signaling:

  • Studies using CNTFR antibodies have revealed that:

    • CNTF must first bind to the alpha component (CNTFR)

    • This permits recruitment of gp130 and LIFR beta to form the tripartite receptor complex

    • Signal transduction through this complex stimulates gene expression, cell survival, and differentiation in neuronal cell types

• Engineered Therapeutics Development:

  • Anti-CNTFR antibodies are essential for validating engineered CLCF1 variants

  • These include:

    • Superagonists with enhanced CNTFR affinity that promote axon regeneration

    • Antagonists that bind CNTFR with high affinity but do not engage coreceptors

  • These engineered molecules may provide novel treatment approaches for neurodegenerative conditions

• Microglial Activation in Neuroinflammation:

  • CNTFR antibodies help distinguish direct effects of CNTF on microglia from indirect effects

  • Studies show CNTF can induce microglial changes similar to IL-6, but with distinct functional outcomes

  • This understanding helps target specific neuroinflammatory mechanisms in disease settings