B'ZETA Antibody

What is the function of IKB zeta in inflammatory signaling pathways?

IKB zeta (encoded by NFKBIZ) is a critical regulator of NF-kappa-B transcription factor complexes with dual inhibitory and activating functions. IKB zeta inhibits NF-kappa-B activity without affecting its nuclear translocation upon stimulation. Specifically, it inhibits DNA-binding of RELA and NFKB1/p50, affecting both the NF-kappa-B p65-p50 heterodimer and the NF-kappa-B p50-p50 homodimer . Interestingly, IKB zeta can also activate NF-kappa-B-mediated transcription in certain contexts. When associated with NFKB1/p50, it exhibits transcriptional activation activity and is recruited to specific promoters such as LCN2 . This protein is particularly involved in the induction of inflammatory genes activated through TLR/IL-1 receptor signaling and plays a role in T helper 17 (Th17) cell differentiation following T cell receptor (TCR) activation .

How does CD3 zeta contribute to T cell receptor signaling?

CD3 zeta, encoded by the CD247 gene, is a 164-amino acid protein belonging to the CD3Z/FCER1G family with a membrane-associated cellular localization . CD3 zeta serves as a critical component of the T cell receptor (TCR) complex, playing an essential role in signal transduction following antigen recognition. The phosphorylation of tyrosine residues within CD3 zeta's immunoreceptor tyrosine-based activation motifs (ITAMs) initiates downstream signaling cascades leading to T cell activation. Specifically, phosphorylation at tyrosine positions such as Y83 and Y142 serves as docking sites for ZAP-70 and other signaling molecules, connecting antigen recognition to cellular responses .

What is the significance of CD20 as a target for scFvFc:zeta receptor-expressing T cells?

CD20 has been validated as an effective target epitope for T cells expressing CD20-specific scFvFc:zeta chimeric receptors. Research has demonstrated that the specific interaction between CD20 molecules on target cells and scFvFc:zeta receptors on engineered T cells triggers functional responses, including cytokine production (IL-2) and cytolytic activity . This interaction is highly specific, as evidenced by the absence of responses when using mock-transfected cells or CD20-negative stimulator cells. Furthermore, the addition of soluble anti-CD20 monoclonal antibodies inhibits this interaction, confirming the specificity of the CD20-scFvFc:zeta binding . These findings have significant implications for adoptive T cell therapies targeting B-cell malignancies expressing CD20.

How does IKB zeta regulate interleukin expression in keratinocytes?

IKB zeta plays a critical regulatory role in the synergistic induction of interleukins by IL-17A and TNFα in human keratinocytes. Recent research has demonstrated that IKB zeta functions as a key regulator of IL-17/TNFα-inducible psoriasis-associated genes and proteins . Specifically, IKB zeta regulates the IL-17A/TNFα-mediated synergistic induction of IL-19 and IL-20, which are important members of the IL-10 cytokine family involved in inflammatory processes . This regulatory mechanism has significant implications for understanding inflammatory skin conditions like psoriasis and potentially developing targeted therapeutic approaches.

What are the optimal validation methods for confirming specificity of anti-CD3 zeta antibodies?

Validation of anti-CD3 zeta antibodies requires a multi-faceted approach to confirm specificity across different experimental contexts:

Western Blot Validation: When validating anti-CD3 zeta antibodies by Western blot, researchers should look for bands at the expected molecular weight (approximately 16-18 kDa for monomeric CD3 zeta). For chimeric constructs like scFvFc:zeta, validation includes confirming the expected size of the chimeric protein (approximately 66 kD under reducing conditions and 132 kD under non-reducing conditions) . Multiple cell types should be tested, including positive controls (T lymphocytes) and negative controls (non-lymphoid cells).

Flow Cytometry Validation: For surface expression analysis, researchers should compare staining patterns between T cells (positive) and non-T cells (negative). For chimeric receptors, dual staining with antibodies recognizing different components (e.g., anti-mouse Fab-specific antibody and anti-human Fc gamma-specific monoclonal antibody) can confirm proper expression and folding .

Functional Validation: Most critical is functional validation through stimulation assays, where antibody binding should trigger expected downstream events. For example, with scFvFc:zeta constructs, coculture with CD20+ target cells should result in measurable IL-2 production or cytolytic activity that is not present with CD20- cells or mock transfectants .

Phosphorylation-Specific Validation: For phospho-specific anti-CD3 zeta antibodies (like those targeting pY83 or pY142), validation should include treatment with phosphatase inhibitors versus phosphatase treatment to confirm specificity for the phosphorylated epitope .

How does antibody developability assessment impact early-stage research decisions?

Antibody developability assessment during early research stages significantly influences candidate selection and optimization strategies. High-throughput (HT) developability screening of 152 human or humanized monoclonal antibodies revealed critical correlations between biophysical properties and downstream process parameters .

Effective developability assessment requires:

Integrated Analysis: The implementation of integrated HT developability workflows at the beginning of antibody discovery campaigns enables researchers to evaluate candidates across multiple parameters simultaneously, including sequence attributes, functional epitope diversity, and biophysical characteristics .

Iterative Optimization: Circular optimization workflows are essential, where analytical characterization informs sequence engineering (e.g., mutagenesis to remove post-translational modifications or disrupt hydrophobic patches), followed by re-analysis to confirm improvements in biophysical properties .

Predictive Modeling: Experimental biophysical data can be used to develop quantitative structure-property relationship (QSPR) models that predict key properties like hydrophobic interaction chromatography (HIC) retention times, which correlate with developability risks .

This approach enables researchers to eliminate antibodies with suboptimal properties early in the selection process and prioritize candidates for further evaluation, ultimately reducing development risks and ensuring only robust antibody molecules progress to later development stages.

What molecular mechanisms underlie IKB zeta's dual regulatory functions?

IKB zeta exhibits both inhibitory and activating functions through distinct molecular mechanisms:

Inhibitory Mechanisms:

• Direct binding to NF-κB subunits, preventing their association with DNA

• Competition with coactivators for binding to NF-κB complexes

• Recruitment of corepressors to specific promoter regions

Activating Mechanisms:

• Formation of activating complexes with NFKB1/p50 that are recruited to specific promoters like LCN2

• Promotion of transcription for genes such as LCN2 and DEFB4

• Recruitment to IL-6 promoters to activate IL-6 expression while simultaneously decreasing TNF-alpha production in response to lipopolysaccharide stimulation

This functional duality appears to be context-dependent, influenced by:

• The specific target gene's promoter architecture

• Cell type-specific cofactors

• The nature of the stimulating signal (e.g., TLR/IL-1 receptor vs. TCR signaling)

• Post-translational modifications of IKB zeta itself

Understanding these mechanisms provides insight into how IKB zeta functions as a critical regulator in both inflammatory responses and Th17 cell differentiation.

What are the optimal experimental designs for studying CD3 zeta-mediated signaling in engineered T cells?

When designing experiments to study CD3 zeta-mediated signaling in engineered T cells (such as those expressing scFvFc:zeta chimeric receptors), researchers should implement the following experimental design elements:

Cell Line Selection:

• Use well-characterized T cell lines (e.g., Jurkat) for initial mechanistic studies

• Validate findings in primary human T cells to confirm physiological relevance

• Include appropriate control cell lines (mock-transfected or expressing irrelevant receptors)

Stimulation Protocols:

• Compare antigen-specific stimulation (e.g., CD20+ target cells for CD20-specific scFvFc:zeta) with non-specific stimulation (e.g., PMA/ionomycin)

• Include titration of target cells to establish dose-response relationships

• Implement time-course experiments to capture both early (minutes) and late (hours) signaling events

Readout Selection:

Validation Strategies:

• Use soluble blocking antibodies to confirm specificity of receptor-target interactions

• Implement genetic approaches (siRNA, CRISPR) to confirm the role of specific signaling molecules

• Compare wildtype and mutant CD3 zeta constructs (e.g., Y83F, Y142F) to dissect the contribution of specific phosphorylation sites

This comprehensive experimental design allows for robust characterization of CD3 zeta-mediated signaling while controlling for potential confounding factors.

How should researchers design experiments to study IKB zeta regulation of IL-17A/TNFα-induced inflammatory responses?

To effectively study IKB zeta's role in regulating IL-17A/TNFα-induced inflammatory responses, researchers should implement the following experimental design strategies:

Cell System Selection:

• Primary human keratinocytes represent an optimal model for studying inflammatory skin conditions

• Compare with other cell types (fibroblasts, immune cells) to establish tissue specificity

• Consider 3D skin equivalents for more physiologically relevant models

Stimulation Conditions:

• Test IL-17A and TNFα both independently and in combination to identify synergistic effects

• Include dose-response and time-course experiments to capture the dynamics of IKB zeta induction

• Compare with other inflammatory stimuli (e.g., IL-1β, LPS) to determine pathway specificity

Gene Expression Analysis:

• Employ RNA-seq or targeted qRT-PCR panels focusing on inflammatory cytokines (IL-19, IL-20)

• Use chromatin immunoprecipitation (ChIP) to identify direct IKB zeta binding to target gene promoters

• Implement nascent RNA analysis techniques to distinguish between transcriptional and post-transcriptional effects

Genetic Manipulation Approaches:

• Use siRNA or CRISPR-Cas9 to knockdown/knockout IKB zeta

• Employ inducible overexpression systems to assess IKB zeta sufficiency

• Generate domain mutants to identify critical regions for different functions

Readout Selection:

This experimental design provides a comprehensive framework for dissecting the complex role of IKB zeta in inflammatory responses triggered by IL-17A and TNFα.

How can researchers address non-specific binding issues with anti-zeta antibodies?

Non-specific binding is a common challenge when working with anti-zeta antibodies. Researchers can implement the following troubleshooting strategies:

Optimization of Blocking Conditions:

• Test multiple blocking agents (BSA, milk, commercial blockers) at various concentrations

• Extend blocking time to ensure complete coverage of non-specific binding sites

• Consider the addition of detergents (0.05-0.1% Tween-20) to reduce hydrophobic interactions

Antibody Dilution Optimization:

• Perform titration experiments to identify the optimal antibody concentration

• For anti-CD3 zeta antibodies, typical dilutions range from 1:500 to 1:2000 for Western blotting

• For flow cytometry applications, concentrations between 1-10 μg/ml are typically effective

Sample Preparation Refinements:

• For cell lysates, ensure complete denaturation for Western blotting applications

• For fixed cells (immunocytochemistry/immunohistochemistry), optimize fixation time and permeabilization methods

• When possible, use phosphatase inhibitors to preserve phosphorylated epitopes

Validation with Multiple Approaches:

• Confirm binding specificity using knockout/knockdown samples as negative controls

• Employ peptide competition assays to verify epitope specificity

• Correlate results across multiple detection methods (e.g., Western blot and immunofluorescence)

Pre-adsorption Strategies:

• Pre-adsorb antibodies with cell lysates from tissues lacking the target protein

• Use recombinant protein columns to purify antibodies when high specificity is required

• Consider using Fc receptor blocking reagents when working with samples containing immune cells

Implementing these strategies systematically can significantly reduce non-specific binding issues and improve data quality when working with anti-zeta antibodies.

What factors should be considered when interpreting contradictory results between different anti-zeta antibody clones?

When faced with contradictory results between different anti-zeta antibody clones, researchers should systematically evaluate several key factors:

Epitope Differences:

• Map the epitopes recognized by each antibody clone

• For CD3 zeta, antibodies targeting different phosphorylation sites (e.g., Y83 versus Y142) may yield different results depending on the activation state of the cells

• For IKB zeta, antibodies recognizing different regions (N-terminal versus C-terminal) may detect different isoforms or post-translationally modified variants

Antibody Format Variations:

• Consider differences between monoclonal and polyclonal antibodies

• Evaluate potential effects of conjugation (fluorophores, enzymes) on epitope recognition

• Assess differences in host species and isotype that may affect non-specific binding

Application-Specific Considerations:

Technical Validation Approaches:

• Use genetic knockout/knockdown models to confirm specificity

• Perform peptide competition assays with specific and non-specific peptides

• Validate with orthogonal methods (e.g., mass spectrometry)

• Test alternative antibody clones from different manufacturers

Biological Context Evaluation:

• Consider cell type-specific differences in target protein expression and modification

• Evaluate the impact of activation state on epitope accessibility

• Assess potential interactions with other proteins that might mask specific epitopes