CHRND Antibody

What is CHRND and why is it significant in research?

CHRND is the delta subunit of the nicotinic acetylcholine receptor found primarily in muscle tissue. It plays a critical role in neuromuscular signal transmission through the acetylcholine receptor (AChR). After binding acetylcholine, the AChR undergoes an extensive conformational change affecting all subunits, leading to the opening of an ion-conducting channel across the plasma membrane . This protein is significant in research because it's implicated in several neurological conditions, particularly myasthenia gravis, where it serves as a main target self-antigen .

What applications are CHRND antibodies typically used for?

CHRND antibodies are versatile research tools applicable across multiple experimental techniques. Based on catalog information from multiple suppliers, these antibodies are validated for:

• Western Blotting (WB)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Flow Cytometry (FACS)

• Immunohistochemistry (IHC) for both frozen and paraffin-embedded tissues

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Immunoprecipitation (IP)

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

How should I select the appropriate CHRND antibody for my specific research application?

When selecting a CHRND antibody, consider:

• Target epitope: Different antibodies target different amino acid sequences (e.g., aa 22-245, aa 175-204, or aa 1-250) . Choose based on which domain of CHRND is relevant to your research.

• Host organism: Available options include mouse monoclonal and rabbit polyclonal . Consider potential cross-reactivity issues, especially when studying mouse samples with mouse-derived antibodies.

• Species reactivity: Verify the antibody has been validated in your species of interest. Some CHRND antibodies work across multiple species (human, rat, mouse, etc.), while others are human-specific .

• Application validation: Ensure the antibody is validated for your specific application. For example, if performing immunohistochemistry on paraffin sections, confirm the antibody works in this context .

• Clonality requirements: For detecting specific epitopes or for quantitative analysis, monoclonal antibodies may be preferred. For maximum sensitivity in detecting native proteins, polyclonal antibodies might be better .

What are the optimal experimental conditions for using CHRND antibodies in different techniques?

Based on the search results, here are recommended conditions:

For Western Blotting:

• Dilution ranges: 1:500-1:5000

• Under non-reducing conditions, the delta subunit may migrate mostly as a dimer of ~130 kDa

• Expected molecular weight: ~59-65 kDa for the delta subunit

For Immunohistochemistry:

• Recommended dilution: 1:1000

• For optimal results with certain antibodies, cryostat sections or permeabilization is recommended due to the reactivity with the cytoplasmic side of the receptor

• When using mouse antibodies on mouse tissue, additional Mouse-on-Mouse blocking steps may be required

For Immunocytochemistry/Immunofluorescence:

• Dilution ranges: 1:20-1:100 or 0.25-2 μg/mL depending on the antibody

• Fixation with formaldehyde prior to staining is recommended for cultured cells

For Flow Cytometry:

• Recommended dilution: approximately 1:200

How can I validate the specificity of my CHRND antibody in experimental settings?

To validate CHRND antibody specificity:

• Positive and negative controls: Use tissues/cells known to express CHRND (e.g., skeletal muscle at motor endplates) versus those that don't .

• Knockdown/knockout validation: Compare staining between wild-type samples and those where CHRND expression has been reduced or eliminated.

• Pre-absorption test: Pre-incubate the antibody with purified CHRND protein or the immunizing peptide before staining to confirm specific binding.

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of CHRND to confirm consistent localization patterns.

• Expected molecular weight confirmation: For Western blots, verify that the detected band appears at the expected molecular weight (~59-65 kDa for the delta subunit, or ~130 kDa dimer under non-reducing conditions) .

• Cross-reactivity assessment: Some CHRND antibodies (like clone 88B) may detect both gamma and delta subunits in certain species (e.g., Torpedo) but only the delta subunit in others (mouse, rat) .

How should I interpret differences in CHRND antibody binding patterns across tissues?

When interpreting differential binding patterns:

• Tissue-specific expression levels: CHRND is primarily expressed at neuromuscular junctions. Strong staining should be expected at motor endplates in skeletal muscle . Unexpected staining in other tissues should be carefully validated.

• Isoform variations: Consider that different splice variants or post-translational modifications might affect antibody binding. This is particularly relevant when comparing embryonic versus adult tissues, as receptor subunit composition changes during development.

• Cross-reactivity assessment: Some antibodies show cross-reactivity with related proteins. For example, the 88B clone detects both gamma and delta subunits in torpedo but only the delta subunit in mouse and rat samples .

• Subcellular localization: CHRND should primarily localize to the cell membrane at synaptic junctions. Unexpected cytoplasmic or nuclear staining may indicate non-specific binding or altered receptor trafficking in pathological conditions.

• Species differences: Interpret results within the context of the species being studied. For example, search results indicate that antibodies may show different binding patterns across human, rat, and Torpedo samples .

What challenges might affect the detection of CHRND in experimental samples?

Several factors can challenge CHRND detection:

• Receptor conformation: Nicotinic acetylcholine receptors undergo conformational changes upon binding acetylcholine . Some antibodies may be conformation-specific, affecting detection depending on receptor state.

• Sample preparation effects: For IHC, some antibodies require specific preparation. For example, the 88B clone recommends cryostat sections or permeabilization due to its reactivity with the cytoplasmic side of the receptor .

• Cross-reactivity with other nAChR subunits: Some antibodies may cross-react with other acetylcholine receptor subunits, particularly between gamma and delta subunits which share structural similarities .

• Mouse-on-Mouse background: When using mouse-derived antibodies on mouse tissue, additional blocking steps may be required to reduce background signal .

• Receptor density variations: The density of acetylcholine receptors varies across different regions of the neuromuscular junction and in pathological conditions, affecting signal intensity.

How can I determine if my CHRND antibody results correlate with clinical manifestations of neuromuscular disorders?

Recent research shows complex relationships between antibody detection and clinical manifestations:

• Correlation analysis approach: Studies examining the relationship between acetylcholine receptor antibody concentrations and clinical symptoms (e.g., in myasthenia gravis) have shown inconsistent correlations. For example, one systematic retrospective study involving 67 patients found that AChR-Ab concentration correlated with MGFA Classification in only 29.4% of patients in the pyridostigmine-only group .

• Clinical scoring systems: Use standardized clinical assessment tools such as the Myasthenia Gravis Foundation of America (MGFA) Clinical Classification, Quantitative Myasthenia Gravis (QMG) score, and MG-specific activities of daily living (MG-ADL) scoring systems to evaluate correlations .

• Statistical analysis methods: Apply Spearman correlation analysis to determine the relationship between antibody concentrations and clinical scores .

• Monitoring longitudinal changes: Track changes in antibody concentration and clinical score over time using MGFA-antibody concentration–treatment plots to assess treatment effects .

• Patient stratification: Consider stratifying patients based on treatment regimens (e.g., immunosuppressive therapy versus pyridostigmine only) when analyzing correlations between antibody detection and symptoms .

How are CHRND antibodies being utilized in cell-based assays for autoimmune disorder diagnosis?

Recent research has explored advanced applications of nAChR antibodies:

A 2024 study developed cell-based assays (CBAs) to detect potentially pathogenic antibodies to neuronal nAChR subtypes in autoimmune encephalitis syndromes (AES) . Key methodological approaches included:

• Live cell-based assay development: Researchers created a sensitive live-CBA with α4β2- or α7-nAChR-transfected cells to detect antibodies against extracellular domains of nAChR major subunits .

• Flow cytometry confirmation: FACS was performed to validate CBA findings and quantitatively confirm the presence of specific antibodies .

• Tissue binding validation: Indirect immunohistochemistry (IHC) was used to investigate serum autoantibodies' binding to rat brain tissue, specifically in cerebellum and hippocampus regions .

• Cross-reactivity elimination: Controls ensured no serum antibodies bound to control-transfected cells and no control serum antibodies bound to the transfected cells .

• Clinical correlation: The study found that patients positive for serum anti-nAChRs α4 subunit antibodies (but negative for other AES-associated antibodies) fell into the AES spectrum with conditions including Rasmussen encephalitis, autoimmune meningoencephalomyelitis, and possible autoimmune encephalitis .

What is the role of CHRND gene polymorphism in autoimmune disorders like myasthenia gravis?

Research has identified interesting genetic associations:

• Microsatellite repeat analysis: Studies using a microsatellite repeat located in the second intron of the CHRND gene observed preferential transmission of the allele 268 in 114 one-generation families with one myasthenic child (Pc=0.0154) .

• Patient group stratification: This allele was over-represented in 350 unrelated nonthymoma MG patients (OR=1.78, P=0.038), but not in 84 thymoma patients, compared to 168 healthy controls .

• Antibody-based subgrouping: Among nonthymoma patients, those lacking serum anti-titin antibodies showed stronger association (OR=2.07, P=0.017) .

• SNP transmission analysis: Interestingly, there was no distortion in the transmission of single-nucleotide substitution polymorphisms (SNPs) in the 3' untranslated region of CHRND nor in that of two SNPs located in the closely linked CHRNG gene, 4.5 kb telomeric to CHRND .

• Research implications: These findings suggest that specific polymorphisms in the CHRND gene may contribute to susceptibility to certain subtypes of myasthenia gravis, warranting detailed investigation of CHRND polymorphism in MG patients .

How can researchers optimize CHRND antibody-based detection methods for studying receptor trafficking and degradation?

To optimize detection methods for receptor dynamics:

• Pulse-chase experiments: Design experiments using temporally controlled antibody labeling to track the lifecycle of CHRND-containing receptors from synthesis through endocytosis and degradation.

• Live-cell imaging: Utilize fluorescently conjugated anti-CHRND antibody fragments (Fab) that don't cross-link receptors to visualize receptor movement in real time.

• pH-sensitive fluorescent tags: Incorporate pH-sensitive fluorophores on anti-CHRND antibodies to distinguish surface receptors from those in acidic endosomal compartments.

• Epitope accessibility considerations: Select antibodies targeting extracellular domains (e.g., aa 22-245 ) for surface receptor detection and those targeting intracellular domains for internalized receptor pools.

• Quantitative analysis approaches: Implement high-content imaging systems with automated analysis algorithms to quantify changes in receptor distribution across cellular compartments under different experimental conditions.

What are the latest developments in using CHRND antibodies for studying neuromuscular junction formation and maintenance?

Recent advances have expanded our understanding of neuromuscular junctions:

• Synaptogenesis studies: CHRND antibodies are being used to track acetylcholine receptor clustering during early stages of neuromuscular junction formation, particularly in developmental models.

• Agrin-MuSK-Rapsyn signaling: Researchers are utilizing anti-CHRND antibodies to study how this pathway regulates AChR clustering at the post-synaptic membrane.

• Disease model applications: In models of neuromuscular disorders, CHRND antibodies help visualize changes in receptor density and distribution patterns that correlate with disease progression.

• Pharmacological intervention assessment: These antibodies are valuable for evaluating how therapeutic compounds affect receptor stabilization at the neuromuscular junction in conditions like myasthenia gravis.

• Regeneration research: Following nerve injury, CHRND antibodies allow visualization of receptor re-clustering during reinnervation and functional recovery of the neuromuscular junction.

What are the optimal storage conditions for maintaining CHRND antibody stability and performance?

For optimal CHRND antibody preservation:

• Temperature requirements: Store at -20°C long term . Some antibodies may be stored at 4°C for short-term use .

• Aliquoting recommendations: To prevent repeated freeze-thaw cycles, aliquot antibodies into single-use volumes before freezing .

• Preservative considerations: Many CHRND antibodies contain preservatives like sodium azide (e.g., 0.05%) to prevent microbial growth. Note that sodium azide is incompatible with some applications, particularly those involving horseradish peroxidase.

• Formulation variations: Antibodies may be supplied in different formulations, including ascites or as purified antibodies in buffers like PBS (pH 7.2) with 40% glycerol . Storage recommendations may vary based on formulation.

• Stability monitoring: Periodically verify antibody performance against positive controls if stored for extended periods.