nbas Antibody

What is NBAS and why is it important in antibody research?

NBAS encodes a protein that forms part of the Syntaxin 18 complex involved in Golgi-to-ER retrograde transport. Mutations in NBAS have been associated with multiple phenotypes including fever-triggered recurrent acute liver failure, short stature, and immune system abnormalities.

In experimental contexts, NBAS protein detection is critical for studying:

• The pathophysiology of NBAS-associated diseases

• Protein expression levels in patient cells

• Protein-protein interactions in vesicular transport

Research has demonstrated that NBAS-deficient patients show consistent immunological abnormalities including reduced CD56+ NK cells, resulting in >50% lower absolute NK cell numbers relative to healthy controls . This makes NBAS an important target for immunological research.

What methods are available for NBAS antibody validation?

Validating NBAS antibodies requires a systematic approach following established principles for research antibody validation. Based on enhanced validation guidelines, researchers should employ at least two of the following methods:

• Genetic validation: Testing antibody reactivity in wild-type versus NBAS knockout or knockdown models. This represents the gold standard for specificity validation .

• Orthogonal validation: Comparing antibody-based protein detection with antibody-independent methods (e.g., mass spectrometry) .

• Independent antibody validation: Testing multiple antibodies targeting different epitopes of NBAS and comparing their staining patterns .

• Expression validation: Testing the antibody in recombinant expression systems where NBAS is overexpressed compared to control cells .

• Capture mass spectrometry: Immunoprecipitating NBAS and confirming its identity through mass spectrometry .

A comprehensive validation study by Edfors et al. demonstrated that combining these approaches significantly improves antibody reliability, with 1,630 antibodies validated by at least two pillars and 267 validated by three or more pillars .

How should I design proper controls for NBAS antibody experiments?

Research by Rimm (2010) emphasizes that peptide controls alone are particularly unreliable, as the lack of signal only demonstrates binding to the immunogen but does not exclude cross-reactivity with unrelated proteins .

What sample preparation methods are optimal for NBAS antibody applications?

When preparing samples for NBAS antibody detection, consider these methodological approaches:

• For Western blot applications:

  • Cells should be collected, washed in PBS, and resolved in RIPA buffer

  • Approximately 10 μg of protein per sample should be separated on a 4%-12% acrylamide gradient gel

  • Primary antibodies against NBAS should be incubated overnight (typical dilution 1:2,000)

  • Enhanced chemiluminescence detection is recommended for protein visualization

  • Protein levels should be quantified with appropriate software such as Bio-1D

• For immunohistochemistry applications:

  • Tissue fixation conditions should be optimized and standardized across experiments

  • Antigen retrieval methods may be necessary and should be validated

  • Always include NBAS knockout or knockdown tissues as negative controls

  • Consider dual staining with other markers to establish co-localization patterns

Research by Gurgor et al. (2016) emphasized the importance of knockout controls in immunohistochemistry applications to ensure reliable antibody validation .

How can I distinguish between neutralizing and non-neutralizing NBAS antibodies?

While specific information about neutralizing versus non-neutralizing NBAS antibodies is limited, general principles from antibody research can be applied. Based on studies of other proteins:

• Functional assays: Develop cell-based assays that measure NBAS activity. If the antibody inhibits this activity, it may be neutralizing.

• Competition assays: Design competition-based ELISA approaches to determine if antibodies prevent protein-protein interactions critical for NBAS function .

• Epitope mapping: Identify if antibodies bind to functional domains of NBAS versus non-functional regions. Research on factor VIII (FVIII) antibodies shows that neutralizing antibodies typically target functional epitopes, while non-neutralizing antibodies often bind to non-functional epitopes .

• Isotype and subclass analysis: Evidence from hemophilia research indicates that IgG4 subclass antibodies correlate strongly with neutralizing activity. In a comprehensive study of 788 individuals, IgG4 subclass antibodies were only observed in persons with a history of inhibitor development .

For precise classification, consider developing a Nijmegen ultra-sensitive assay approach that can detect even very low-titer neutralizing antibodies (down to 0.10 units/mL) as demonstrated for FVIII research .

What are the optimal methods for quantifying NBAS antibody levels in patient samples?

For accurate quantification of NBAS antibodies in research samples, consider these methodological approaches:

• High-dynamic range fluorescent bead-based immunoassay:

  • Immobilize biotinylated NBAS protein on streptavidin beads

  • Detect specific antibodies using anti-Ig (IgG, IgA, IgM, IgG1-4) specific secondary antibodies conjugated to fluorescent tags

  • Analyze using flow cytometry for superior dynamic range compared to ELISA

  • This approach has demonstrated high sensitivity (down to picogram ranges) for detecting antibodies against other proteins

• Competitive ELISA with recombinant protein:

  • Develop an assay based on the competition between patient antibodies and a known binding partner of NBAS

  • This approach has shown high specificity (99.93%) and sensitivity (95-100%) in SARS-CoV-2 antibody detection

• Multi-parametric flow cytometry:

  • For detecting cell-associated NBAS, implement panels similar to those used for immune cell characterization

  • Panel design should include multiple parameters (≥8) for comprehensive characterization

  • Use appropriate backbone markers for absolute quantification

  • Include activation markers to assess functional status

Research indicates that bead-based assays provide wider dynamic range compared to traditional ELISA approaches (demonstrated correlation r = 0.86 between methods) .

How does NBAS deficiency affect immune cell populations and antibody responses?

NBAS deficiency has significant effects on immune cell populations that researchers should consider when designing antibody-based studies:

• NK cell abnormalities:

  • NBAS-deficient patients show consistent reduction in CD56+ NK cells

  • Results in >50% lower absolute NK cell numbers relative to healthy controls

  • May affect NK cell activation (CD69 expression) and functional markers (perforin, granzyme B)

• T cell alterations:

  • Changes in CD4+/CD8+ T cell ratios have been observed

  • In one patient study, the proportion of CD4+ T cells decreased while CD8+ T cells increased

• B cell and antibody production:

  • Hypogammaglobulinemia has been reported in >60% of NBAS-deficient patients

  • Abnormal κ-deleting recombination excision circles (KREC) have been detected via newborn screening

These immunological abnormalities suggest that NBAS plays a role in immune system development and function, which may affect antibody production and responses in NBAS-deficient individuals.

What computational approaches can be used to predict NBAS antibody binding epitopes?

Advanced computational methods can help predict potential binding epitopes for NBAS antibodies, which is particularly valuable for designing experiments and interpreting results:

• Biophysics-informed modeling:

  • Developed to identify different binding modes associated with specific ligands

  • Can disentangle multiple binding contributions from a single experiment

  • Allows prediction and generation of specific variants beyond those observed in experiments

• Machine learning approaches:

  • Can classify antibody sequences observed in selection experiments

  • Help extract patterns from non-specific antibodies that bind several targets

  • Useful for designing new, experimentally untried antibody sequences with specific properties

• Structural computation and library generation:

  • Enables screening of properties of antibody and antibody-like molecules

  • Can tailor affinity and stability for improved results

  • Useful in the early discovery phase focusing on antibody binding properties

Research by Pfab et al. demonstrated that combining biophysics-informed modeling with extensive selection experiments has broad applicability beyond specific antibody targets, offering powerful tools for designing proteins with desired physical properties .

How can I optimize NBAS antibody detection in challenging tissue samples?

Detecting NBAS in challenging tissue samples requires advanced methodological approaches:

• Tissue-specific optimization:

  • Different tissues may require distinct fixation and processing protocols

  • Systematic testing of antibody dilutions is essential for optimal signal-to-noise ratio

  • For tissues with high autofluorescence, consider spectral unmixing or alternative detection methods

• Immunograms for complex samples:

  • Implement HPLC-ELISA or LC-ELISA approaches for complex biological samples

  • These techniques can identify unexpected cross-reactants and improve specificity

  • Particularly useful for environmental or inherently complex samples

• Single-cell approaches:

  • For heterogeneous tissues, consider single-cell protein profiling

  • Enables correlation of NBAS expression with cellular phenotypes

  • Mass cytometry (CyTOF) or imaging mass cytometry may provide higher parameter detection than conventional immunofluorescence

• Sensitivity enhancement strategies:

  • Signal amplification using tyramide signal amplification

  • Proximity ligation assays for detecting protein-protein interactions

  • Upconversion of phosphorescent nanoparticles as markers for quantitative detection

Research indicates that the sensitivity of detection methods can vary significantly (28.7%-93.1%), with substantial differences between different protocols and reagents .

How are NBAS antibodies used in researching liver diseases?

NBAS mutations are associated with recurrent acute liver failure, making antibodies against NBAS valuable tools in liver disease research:

• Diagnostic applications:

  • NBAS antibodies enable identification of protein expression patterns in liver biopsies

  • Can help distinguish NBAS-associated liver failure from other causes

  • May be used to correlate NBAS protein levels with disease severity

• Functional studies:

  • NBAS antibodies allow visualization of subcellular localization in hepatocytes

  • Enable assessment of protein-protein interactions critical for ER-Golgi transport

  • Facilitate studies of stress-induced changes in NBAS expression and localization

• Pathophysiological investigations:

  • Help determine if NBAS mutations lead to protein absence, reduction, or dysfunction

  • Testing fibroblasts from affected individuals has shown reduction of NBAS levels to 18%-36% compared to controls

  • Indicates substantial impairment of protein translation and/or stability in affected individuals

Research demonstrated that genetic mutations like c.1533_1545del];[2951T>G] and c.[1042C>T];[2203−3C>G] result in significantly decreased NBAS protein levels, contributing to disease manifestation .

What is the relationship between NBAS antibody detection and clinical immunological findings?

NBAS deficiency has significant immunological manifestations that can be studied using antibody-based approaches:

• Natural Killer (NK) cell abnormalities:

  • Flow cytometry using antibody panels can quantify the reduction in CD56+ NK cells characteristic of NBAS deficiency

  • Functional assays measuring CD107a and CD69 expression can assess NK cell activation

  • Intracellular staining for perforin and granzyme B can evaluate NK cell effector function

• T cell population alterations:

  • Comprehensive antibody panels can characterize naïve and memory/effector T cell populations

  • Research has shown altered CD4+/CD8+ T cell ratios in NBAS-deficient patients

  • Antibodies against specific markers (CD183, CD185, CD196) help identify Th1, Th2, Th17, and Tfh cells

• B cell and antibody production:

  • Flow cytometry panels using CD19, CD20, IgD, CD27, CD10, CD24, and CD38 can identify transitional, naïve, and memory B cells

  • Can help understand the mechanism of hypogammaglobulinemia in NBAS deficiency

  • Enables tracking of circulating plasmablasts in absolute numbers and ratios

Research using these approaches has identified that immunological symptoms occur in more than 60% of patients with NBAS-associated disease across all defined subgroups .

How can longitudinal studies of NBAS antibody levels inform disease progression?

Longitudinal monitoring of NBAS protein levels and associated antibodies can provide valuable insights into disease mechanisms and progression:

• Temporal analysis approaches:

  • Serial sampling at defined intervals following diagnosis

  • Use of consistent detection methods to ensure comparability

  • Statistical approaches like generalized additive models (GAMs) can nonparametrically assess associations between continuous variables

  • Cubic smoothing splines can be used to visualize temporal patterns

• Correlation with clinical parameters:

  • Track NBAS protein levels in relation to liver enzyme fluctuations

  • Monitor during fever episodes to assess temperature-related changes

  • Correlate with growth parameters in patients with short stature phenotype

• Treatment response monitoring:

  • Evaluate changes in NBAS expression following therapeutic interventions

  • Use consistent antibody-based detection methods across timepoints

  • Consider multiple testing correction using methods like Benjamini and Hochberg to control for false discovery rate

Research approaches using these longitudinal methodologies have been successfully applied to study antibody responses in other contexts, revealing important temporal patterns that inform disease mechanisms .

What are common pitfalls in NBAS antibody research and how can they be avoided?

Researchers should be aware of these common methodological challenges when working with NBAS antibodies:

• Cross-reactivity issues:

  • Unwanted and unexpected cross-reactivities are always possible due to the lock-and-key mechanism of antibody-antigen interactions

  • Antibodies may provide identical staining patterns in wild-type and knockout tissues, indicating cross-reactivity with unknown epitopes

  • Comprehensive cross-reactivity testing is essential but often limited by the arbitrary selection of tested cross-reactants

• Batch variation:

  • Different batches of the same antibody may show different properties

  • Lot-to-lot and run-to-run reproducibility testing is crucial

  • New batches of tissue samples might differ in hidden properties and behave differently with some antibodies

• Validation limitations:

  • Inhibition tests or preadsorption experiments with peptide antigens might be insufficient to support claimed selectivity

  • RNA interference (siRNA) assays may not fully eliminate target expression

  • Peptide controls are particularly unreliable for specificity testing

• Detection limitations:

  • For environmental or complex samples, consider using immunograms (HPLC-ELISA and LC-ELISA)

  • These approaches can identify unexpected cross-reactants in complex samples

Research by Rimm (2010) emphasizes that validation should include testing for sensitivity, specificity, and reproducibility to ensure reliable antibody performance .

How can I optimize western blot protocols specifically for NBAS detection?

NBAS is a large protein (~320 kDa), requiring specific optimization for western blot detection:

• Sample preparation:

  • Cells should be collected, washed in PBS, and resolved in RIPA buffer

  • Use protease inhibitors to prevent degradation of the large NBAS protein

  • For consistent results, standardize protein quantification methods

• Gel electrophoresis optimization:

  • Use 4%-12% acrylamide gradient gels for optimal separation

  • Load approximately 10 μg of protein per sample

  • Consider longer running times to properly resolve high molecular weight proteins

• Antibody application:

  • Primary antibodies against NBAS should be incubated overnight (typical dilution 1:2,000)

  • Include β-actin (1:15,000) as loading control

  • Optimize secondary antibody concentrations to minimize background

• Detection and quantification:

  • Enhanced chemiluminescence detection systems like Vilberscan Fusion FX7 provide good sensitivity

  • Quantify protein levels with appropriate software (e.g., Bio-1D)

  • Express NBAS levels as a percentage relative to controls

Research studying NBAS mutations showed that affected individuals had reduction of NBAS levels to 18%-36% compared to controls, demonstrating the importance of accurate quantification .

How should I approach the development of novel NBAS antibodies for research?

When developing new NBAS antibodies for research applications, consider these methodological approaches:

• Target selection:

  • Identify functional domains or unique epitopes within NBAS

  • Consider evolutionary conservation for cross-species applications

  • For high specificity, target regions with minimal homology to other proteins

• Antibody generation strategies:

  • Phage display technology can generate antibodies against diverse targets

  • Design libraries based on validated human V domains

  • Consider libraries where key complementary determining regions (CDRs) are systematically varied

• Screening and selection:

  • Implement multiple rounds of selection with appropriate controls

  • Collect phages at each step to monitor antibody library composition

  • High-throughput sequencing can provide comprehensive characterization of selected antibodies

• Biophysics-informed modeling:

  • Build computational models to express probability of antibody selection

  • Define binding modes mathematically with parameters that depend on both the experiment and the sequence

  • Use optimization approaches to design novel sequences with predefined binding profiles

Research by Pfab et al. demonstrated that biophysically interpretable models can disentangle different contributions to binding from a single experiment, allowing researchers to design new antibody sequences that discriminate closely related ligands .

How do I select the most appropriate NBAS antibody for my specific research application?

Selecting the optimal NBAS antibody requires careful consideration of your experimental context:

• Application-specific validation:

  • Antibodies validated for western blot may not perform in immunohistochemistry

  • Enhanced validation should be specific for a certain sample context

  • Consider that validation depends on the sample preparation procedures used

• Target epitope considerations:

  • For detecting specific NBAS variants, select antibodies targeting conserved regions

  • For distinguishing between variants, choose antibodies that recognize unique epitopes

  • Consider accessibility of epitopes in native versus denatured conditions

• Performance metrics assessment:

  • Review validation data across multiple methods (orthogonal, genetic, independent antibody)

  • Prioritize antibodies validated by multiple pillars (e.g., 267 antibodies in Edfors et al. study were validated by three or more methods)

  • Consider sensitivity and specificity trade-offs based on your application needs

• Experimental controls planning:

  • Ensure appropriate positive and negative controls are available

  • For highest confidence, prioritize antibodies with knockout validation data

  • Consider if tissue-specific or cell-type-specific controls are needed