NFD5 Antibody

What are Neurofascin antibodies and what epitopes do they recognize?

Neurofascin antibodies are autoantibodies that target different isoforms of the Neurofascin protein, which plays critical roles in axon-glial interactions at the nodes of Ranvier. The main antibodies identified in research settings include anti-NF155, anti-NF186, and anti-NF140, each targeting distinct epitopes on different Neurofascin isoforms. These antibodies can be detected in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and are associated with specific clinical phenotypes .

The detection of these antibodies requires specialized cell-based assays (CBA) and enzyme-linked immunosorbent assays (ELISA), which identify autoantibodies binding to specific domains of the Neurofascin protein. Most research focuses on the NF155 isoform, which is localized to the paranodal junction between axons and myelin sheaths .

What detection methods yield the highest sensitivity for anti-Neurofascin antibodies?

Two complementary methods are recommended for optimal detection of anti-Neurofascin antibodies:

• Cell-Based Assay (CBA): This method utilizes HEK293 cells transfected with GFP-tagged NF155 (or other Neurofascin isoforms). The cells are fixed in phosphate-buffered saline using 4% paraformaldehyde and blocked using 5% bovine serum albumin. Patient sera are incubated with the cells at 1:20 dilution for 1 hour at room temperature, followed by incubation with fluorescently-labeled secondary antibodies. Results are evaluated via fluorescence microscopy by measuring the fluorescence intensity and assessing the overlap between GFP-tagged NF155 and the antibody signal .

• ELISA: This quantitative method provides complementary confirmation. In research published in The Journal of Clinical Neurology, all serum samples positive in CBA were also positive for anti-NF155 antibodies by ELISA, demonstrating the value of using both techniques in parallel .

For research requiring high confidence in results, combining both detection methods is strongly recommended, as this approach minimizes false positives and provides both visual confirmation and quantitative measurement.

What is the prevalence of anti-NF155 antibodies in CIDP patients?

The prevalence of anti-NF155 antibodies varies across different studies and populations. In a recent South Korean study of 68 patients who fulfilled the 2010 EFNS/PNS diagnostic criteria for CIDP, 6 patients (8.8%) were positive for anti-NF155 antibodies by both CBA and ELISA. Previous studies have reported prevalence rates ranging from 4% to 25% in CIDP populations .

This variation may be attributed to:

• Differences in inclusion criteria for subject selection

• Variations in antibody detection methodologies

• Sample size limitations

• Geographic and genetic differences between study populations

The recent study referenced above represents one of the larger cohorts and found that approximately 9% of CIDP patients had anti-NF155 antibodies, which aligns with a large previous study that found these antibodies in approximately 5% of all CIDP patients .

Which immunoglobulin class predominates in anti-NF155 positive patients?

Research consistently shows that IgG4 is the predominant immunoglobulin subclass in patients with anti-NF155 antibodies. In the study of South Korean patients, IgG4 was identified as the predominant subclass in four of the six patients (66.7%) who tested positive for anti-NF155 antibodies .

This predominance of IgG4 has important implications for understanding disease mechanisms and therapeutic approaches. IgG4 antibodies typically do not activate complement but can disrupt protein-protein interactions, which may explain the specific pathological mechanisms in anti-NF155 antibody-associated CIDP and its distinct response pattern to different immunotherapies.

How do anti-NF antibodies mechanistically contribute to demyelination in CIDP?

Anti-NF antibodies, particularly anti-NF155, are thought to disrupt the paranodal axoglial junctions through several mechanisms:

• Disruption of protein interactions: NF155 interacts with the Caspr/contactin complex to maintain paranodal integrity. Anti-NF155 antibodies (predominantly IgG4) interfere with these protein-protein interactions without necessarily triggering complement activation or antibody-dependent cellular cytotoxicity.

• Paranodal dismantling: By disrupting the paranodal architecture, these antibodies compromise the barrier function of paranodes, allowing voltage-gated potassium channels to redistribute from the juxtaparanodal region into the paranodes.

• Impaired saltatory conduction: The structural changes lead to conduction slowing and eventual conduction block, which manifests clinically as progressive neurological deficits.

• T-cell involvement: Research suggests a potential role for T cells specifically reactive to Neurofascin epitopes. Studies have utilized experimental protocols where peripheral blood mononuclear cells are cultured (5 cells/well) with NF155 and NF186 antigens at concentrations of 40 μg/ml, in the presence of anti-CD28 antibody (2 μg/ml) to detect T-cell responses .

These mechanisms collectively contribute to the demyelinating process and the distinct clinical phenotype observed in anti-NF155-positive CIDP patients.

What is the differential response to immunotherapies in anti-NF155 positive versus negative CIDP patients?

Anti-NF155 antibody status significantly influences therapeutic responses in CIDP patients, with important implications for treatment algorithms:

Table 1: Treatment Response Patterns in Anti-NF155 Positive CIDP Patients

This treatment response pattern contrasts with typical CIDP, where approximately 60-80% of patients respond to either corticosteroids or IVIg. The high response rate to rituximab in anti-NF155 positive patients (100% in the studied cohort) suggests that early B-cell targeted therapy should be considered in these patients, particularly after documenting poor response to conventional treatments .

The mechanistic explanation for this differential response likely relates to the predominance of IgG4 antibodies, which are produced by B cells that may be particularly susceptible to rituximab-mediated depletion.

What methodological considerations are critical when designing T-cell response studies to Neurofascin epitopes?

When investigating T-cell responses to Neurofascin epitopes, researchers should implement the following methodological considerations:

• Antigen preparation: Utilize properly folded Neurofascin (NF155 and NF186) antigens at concentrations of approximately 40 μg/ml to effectively stimulate potential T-cell responses.

• T-cell co-stimulation: Include anti-CD28 antibody (2 μg/ml) in the culture medium to provide necessary co-stimulatory signals for T-cell activation.

• Controls:

  • Negative control: Use CTL-Test-Medium to detect spontaneous IFN-γ secretion

  • Positive control: Include CEF peptide pool (containing 23 MHC class I restricted viral antigens) at 10 μg/ml

• Culture conditions: Maintain cultures at 37°C with 5% CO₂ for optimal T-cell viability and response.

• Readout systems: Employ ELISPOT assays for IFN-γ or multiparameter flow cytometry to assess cytokine production profiles and identify responding T-cell subsets.

• Cell density: Plate approximately 5 cells/well when testing for specific T-cell reactivity to maximize the detection of rare antigen-specific T cells .

These methodological details are critical for obtaining reliable and reproducible results when investigating the potential role of T-cell responses in the pathogenesis of anti-NF antibody-associated disorders.

How do the binding characteristics of antibodies targeting different Neurofascin isoforms compare?

The binding characteristics of antibodies targeting different Neurofascin isoforms reveal important distinctions that influence their pathophysiological effects:

Table 2: Comparison of Antibodies Targeting Different Neurofascin Isoforms

While most patients have antibodies targeting a single Neurofascin isoform, the research data shows that some patients can develop antibodies against multiple isoforms. In the referenced study, one of the six patients positive for anti-NF155 antibodies also demonstrated reactivity to both NF186 and NF140 .

This cross-reactivity pattern suggests epitope spreading or recognition of shared domains between different Neurofascin isoforms, which may influence disease progression and treatment response.

What are the optimal experimental conditions for detecting low-titer anti-Neurofascin antibodies?

Detecting low-titer anti-Neurofascin antibodies presents significant technical challenges. Based on methodologies described in current research, the following optimized protocols are recommended:

• Enhanced CBA sensitivity:

  • Use cells with high expression levels of the target protein

  • Reduce serum dilution to 1:10 (compared to standard 1:20)

  • Extend primary antibody incubation time to 2 hours

  • Use high-sensitivity detection systems such as tyramide signal amplification

  • Evaluate results using quantitative fluorescence imaging with background subtraction

• Modified ELISA protocol:

  • Utilize high-binding plates (Thermo Fisher Scientific, 446612)

  • Coat plates with higher concentration of antigen (200 ng/well)

  • Employ more sensitive detection antibodies

  • Use chemiluminescent rather than colorimetric detection

  • Define positivity threshold as mean OD of healthy controls plus 5 standard deviations

• Immunoprecipitation followed by Western blot:

  • For confirmation of borderline positive results

  • Provides additional specificity validation

These methodological enhancements can significantly improve the detection of low-titer antibodies that might be missed by standard protocols, particularly in early disease stages or following immunotherapy when antibody levels may be decreasing .

How do neutralization mechanisms differ between antibodies targeting the N-terminal domain versus other epitopes?

While this question appears more relevant to viral studies than Neurofascin research, the search results provide insights on neutralization mechanisms that may have methodological relevance for researchers:

Studies with SARS-CoV-2 antibodies demonstrate that antibodies targeting different domains (like N-terminal domain versus receptor-binding domain) utilize distinct neutralization mechanisms:

• Post-attachment neutralization: Some antibodies (like SARS2-57 targeting the NTD) retain neutralizing activity when added after virus absorption to cells. This indicates interference with post-attachment steps such as membrane fusion or conformational changes .

• Pre-attachment neutralization: Other antibodies primarily block initial attachment to cellular receptors.

Methodologically, these differences can be studied through:

• Pre- versus post-attachment neutralization assays

• Syncytia formation inhibition tests

• Structural studies using cryo-electron microscopy to determine binding epitopes

For Neurofascin research, similar methodological approaches could potentially be adapted to investigate whether anti-NF antibodies interfere with protein interactions at different stages of cellular processes .

What are the latest developments in humanized antibody models for studying neurological autoantibodies?

Recent developments in humanized antibody models have created new opportunities for studying neurological autoantibodies:

The HuMAb mouse technology, which involves genetic modifications to mouse immunoglobulin loci, allows for the generation of fully human antibodies in mouse models. This approach has been successfully applied to develop potent neutralizing antibodies against viral targets and could be adapted for neurological autoantibody research.

Key methodological aspects include:

• Generation of mice with modified Igh and Igκ loci containing human variable region sequences

• Immunization protocols using properly folded target proteins

• High-throughput antibody repertoire analysis

• Advanced screening methods to identify antibodies with desired binding properties

These humanized mouse models offer significant advantages over traditional approaches by generating antibodies with fully human variable regions that are more relevant for understanding human disease pathogenesis and developing potential therapeutic antibodies .

How can structural biology techniques enhance our understanding of anti-NF antibody epitopes?

Advanced structural biology techniques provide powerful tools for elucidating the precise epitopes recognized by anti-NF antibodies:

• Cryo-electron microscopy (cryo-EM): This technique has been successfully used to determine the structure of antibody-antigen complexes at near-atomic resolution. For example, studies with SARS-CoV-2 antibodies have revealed detailed binding mechanisms that explain variant recognition and immune evasion .

• X-ray crystallography: While not explicitly mentioned in the search results, this technique remains valuable for high-resolution structural determination of antibody-antigen complexes.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can map epitopes by identifying regions of the antigen that are protected from solvent exchange when bound to antibodies.

• Epitope binning using surface plasmon resonance: This method categorizes antibodies based on competitive binding to overlapping epitopes.

Applying these techniques to anti-NF antibodies would provide valuable insights into:

• The precise epitopes recognized by pathogenic antibodies

• Structural determinants of isoform specificity

• Mechanisms of antibody-mediated disruption of protein-protein interactions

• Potential epitope targets for therapeutic intervention

These structural insights would significantly advance our understanding of the pathogenic mechanisms of anti-NF antibodies and guide the development of targeted therapies .

What are the defining clinical characteristics of anti-NF155 antibody-positive patients?

Anti-NF155 antibody-positive patients present with a distinctive clinical phenotype that differentiates them from other CIDP subtypes:

Table 3: Clinical Characteristics of Anti-NF155 Antibody-Positive Patients

The identification of these distinctive features has led to the recognition of anti-NF155 antibody-associated disease as "autoimmune nodopathy," highlighting its unique pathophysiology compared to classical CIDP .

These clinical characteristics underscore the importance of testing for anti-NF155 antibodies in CIDP patients, particularly in younger individuals presenting with pronounced sensory ataxia who show suboptimal response to first-line therapies.

How should researchers design clinical studies to evaluate novel therapies for anti-NF antibody-associated disorders?

Designing effective clinical studies for anti-NF antibody-associated disorders requires careful consideration of several methodological aspects:

• Patient selection:

  • Include validated antibody testing using both CBA and ELISA

  • Stratify by antibody status (anti-NF155, anti-NF186, anti-NF140)

  • Consider IgG subclass analysis for further stratification

• Outcome measures:

  • Use validated CIDP disability scales (e.g., INCAT, ONLS)

  • Include quantitative sensory testing for ataxia evaluation

  • Monitor antibody titers as a biomarker of treatment response

  • Consider electrophysiological parameters (particularly for paranodal dysfunction)

• Study design considerations:

  • Due to the rarity of these conditions, consider multi-center collaboration

  • Implement adaptive trial designs to accommodate small patient populations

  • Use crossover designs when appropriate to increase statistical power

• Treatment selection:

  • Focus on B-cell targeted therapies (e.g., rituximab) based on promising results

  • Consider emerging therapies that specifically target IgG4 antibodies

  • Evaluate combination therapies targeting multiple immune pathways

• Duration and follow-up:

  • Include adequate follow-up (minimum 12 months) to assess sustained response

  • Monitor for relapse following treatment discontinuation

These methodological considerations aim to overcome the challenges of studying rare antibody-mediated disorders while generating robust evidence to guide clinical practice .