NCR3 Antibody, HRP conjugated

What is NCR3 and what is its biological significance?

NCR3 (Natural Cytotoxicity Triggering Receptor 3) is a cell membrane receptor expressed on natural killer (NK) cells that plays a critical role in the innate immune response. This receptor, also known as NKp30 or CD337, functions as an activating receptor that is triggered by binding extracellular ligands including BAG6 and NCR3LG1 . Upon activation, NCR3 stimulates NK cell cytotoxicity toward neighboring cells that produce these ligands, particularly tumor cells, making it an important component of immune surveillance against cancer .

The biological significance of NCR3 extends beyond direct cytotoxicity. Engagement of NCR3 by BAG6 also promotes the maturation of myeloid dendritic cells (DCs) through two mechanisms: killing DCs that have not acquired a mature phenotype, and inducing NK cells to release TNFA and IFNG, which further promote DC maturation . This indicates NCR3's important role in bridging innate and adaptive immunity.

Research has demonstrated that high-affinity antibodies targeting NCR3 can effectively stimulate NK cell-mediated cytotoxicity and interferon-γ secretion, suggesting potential applications in immunotherapeutic approaches . These findings underscore the significance of NCR3 as both a research target and a potential therapeutic avenue.

What are the optimal storage and handling conditions for NCR3 Antibody, HRP conjugated?

For optimal performance of NCR3 Antibody, HRP conjugated, proper storage and handling conditions are essential. The antibody should be stored at -20°C for long-term preservation or at 4°C for shorter periods (up to one month) . The product typically contains preservatives such as 0.03% Proclin 300 and is formulated in a solution of 50% Glycerol with 0.01M PBS at pH 7.4 .

When handling the antibody, it's advisable to:

• Avoid repeated freeze-thaw cycles which can lead to protein degradation and loss of activity

• Aliquot the antibody upon receipt if multiple uses are planned

• Thaw frozen aliquots on ice or at 4°C rather than at room temperature

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

• Maintain sterile conditions when handling the antibody

For dilution purposes, use buffers free of endogenous peroxidase activity when working with HRP-conjugated antibodies to prevent background signal. Additionally, the inclusion of carrier proteins such as BSA (0.1-1%) can help stabilize the antibody in more dilute solutions.

What are the primary applications for NCR3 Antibody, HRP conjugated?

Key applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): The primary recommended application, useful for quantitative detection of NCR3 in samples .

• Immunohistochemistry: Can be used to detect NCR3 expression in tissue sections, enabling spatial analysis of NCR3 distribution in tumors or immune-related tissues.

• Western Blot: Though not specifically recommended for the HRP-conjugated format in the provided information, anti-NCR3 antibodies can be used to detect the protein in cell or tissue lysates.

• Flow Cytometry: Anti-NCR3 antibodies are valuable for analyzing NCR3 expression on NK cells and for sorting NK cell populations .

When using this antibody for experimental applications, researchers should consider performing validation studies to determine optimal dilutions and conditions specific to their experimental systems, as these may vary from the general recommendations provided by manufacturers.

How does antibody affinity for NCR3 correlate with NK cell activation?

Research has revealed a significant correlation between antibody affinity for NCR3 and the ability to stimulate NK cell activity. High-affinity antibodies targeting NCR3 and other activating receptors (such as CD16 and NCR1) are capable of stimulating NK cell-mediated cytotoxicity and interferon-γ secretion, whereas low-affinity antibodies targeting the same receptors often fail to elicit similar responses . This correlation has important implications for both research applications and therapeutic development.

The relationship between affinity and activation appears to be consistent across multiple NK cell activating receptors. For instance, studies have shown that higher-affinity CD16 polymorphisms demonstrate enhanced antibody-dependent cellular cytotoxicity (ADCC) and are associated with improved clinical responses to therapeutic antibodies like rituximab, trastuzumab, and cetuximab . This pattern extends to NCR3, where functional screening has identified that high-affinity binders are more effective at stimulating NK cell activity.

It's important to note that while high affinity is necessary, it is not always sufficient for NK cell activation. Research has found that high-affinity antibodies targeting NK cell receptors beyond known activating receptors (such as costimulatory receptors TNFRSF9 and CD244) were unable to stimulate NK cell-mediated cytotoxicity on their own . This suggests that receptor identity remains critical, and NK cell activation typically requires engagement of specific activating receptors or co-engagement of different activating and costimulatory receptors.

What methodological approaches can optimize NCR3 Antibody use in bispecific antibody development?

Developing bispecific antibodies using NCR3-targeting components requires careful consideration of several methodological approaches to optimize their effectiveness. Based on research findings, the following strategies can enhance bispecific antibody performance:

• Domain ordering optimization: Studies have shown that single-chain variable fragment (scFv) domain ordering significantly impacts the efficacy of NCR3-based bispecific antibodies. In particular, antibodies with variable light chain to variable heavy chain (VL-VH) ordering often outperform those with VH-VL ordering in stimulating NK cell-mediated cytotoxicity . This effect appears consistent across multiple NCR3-targeting antibody clones, suggesting it may be a general principle for NCR3-directed bispecifics.

• Strategic linker attachment: The choice of whether to attach the NK-targeting scFv to the heavy or light chain of the tumor-targeting Fab can significantly influence the bispecific antibody's ability to induce cytotoxicity. Research has demonstrated that these effects are dependent on the specific NK cell-targeting scFv used . For example, in some CD16-based bispecific antibodies, linkage to the light chain of the anti-CD20 Fab proved more effective than linkage to the heavy chain.

• Affinity considerations: Selecting high-affinity NCR3-binding antibodies is critical for developing effective bispecific constructs. Functional screening methods that identify antibodies capable of inducing NK cell-mediated cytotoxicity provide valuable starting materials for bispecific development .

• Target selection compatibility: NCR3-targeting bispecific antibodies have been successfully developed against multiple tumor targets, including CD20 (B cell lymphoma) and HER2 (breast cancer) . This versatility suggests that NCR3-targeting components can be adapted to various tumor-targeting strategies.

When properly optimized, NCR3-targeting bispecific antibodies can be highly effective, with some constructs demonstrating efficacy comparable to or exceeding that of conventional monoclonal antibodies relying on ADCC mechanisms.

How can NCR3 Antibody, HRP conjugated be used to study NK cell-tumor cell interactions?

NCR3 Antibody, HRP conjugated provides valuable tools for investigating the complex interactions between NK cells and tumor cells. Several methodological approaches can leverage this antibody to elucidate these interactions:

• Visualization of receptor-ligand engagement: Using immunohistochemistry or immunofluorescence techniques, researchers can employ NCR3 Antibody to visualize the localization of NCR3 at the NK cell-tumor cell interface, often referred to as the immunological synapse. The HRP conjugation enables sensitive detection in tissue sections or cell culture models, allowing researchers to observe receptor clustering and co-localization with other immune receptors or signaling molecules.

• Quantification of receptor expression levels: Flow cytometry using anti-NCR3 antibodies allows researchers to quantify NCR3 expression levels on NK cells from different tissue sources or under various stimulation conditions. This approach can reveal how tumor microenvironments might modulate NCR3 expression, potentially affecting NK cell activation status.

• Functional blocking studies: By using NCR3 antibodies to block receptor-ligand interactions, researchers can assess the contribution of this specific pathway to NK cell-mediated tumor recognition and killing. Comparing results with and without NCR3 blockade can determine the relative importance of this receptor in different tumor models.

• Monitoring NK cell activation: NCR3 engagement leads to downstream signaling events including cytokine production. Using NCR3 Antibody in combination with assays measuring interferon-γ or TNF-α production can provide insights into how tumor cells trigger NK activation through this receptor pathway .

• Developing improved immunotherapeutic strategies: The understanding gained from studying NCR3-mediated NK-tumor interactions can inform the development of more effective bispecific antibodies. Research has demonstrated that high-affinity antibodies targeting NCR3 can be reformatted into bispecific constructs that effectively redirect NK cell cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells .

What are common challenges when using NCR3 Antibody, HRP conjugated in ELISA and how can they be addressed?

When using NCR3 Antibody, HRP conjugated in ELISA systems, researchers may encounter several technical challenges that can affect assay performance. Understanding these potential issues and their solutions can help optimize experimental outcomes:

• High background signal: This common problem can result from several factors:

  • Solution: Optimize blocking conditions using 1-5% BSA or non-fat dry milk in PBS or TBS buffer. Increasing the concentration or duration of blocking can reduce non-specific binding.

  • Solution: Include 0.05-0.1% Tween-20 in wash buffers and dilute antibody in buffer containing 0.05% Tween-20 to reduce hydrophobic interactions.

  • Solution: Ensure all buffers used are free from endogenous peroxidase activity that could react with the HRP substrate.

• Weak or absent signal:

  • Solution: Verify antibody activity by testing different dilutions below the recommended range. Titrate from 1:100 to 1:10,000 to identify optimal concentration.

  • Solution: Consider alternate capture antibodies with different epitope recognition to improve detection sensitivity.

  • Solution: Extend substrate incubation time but monitor closely to prevent signal saturation.

  • Solution: Confirm target protein expression in your sample source, as NCR3 expression can vary across cell types and activation states.

• Cross-reactivity issues:

  • Solution: The NCR3 Antibody is specifically reactive with human NCR3 , so validate sample species compatibility.

  • Solution: Perform pre-absorption controls with recombinant NCR3 protein to confirm signal specificity.

  • Solution: Include appropriate negative controls lacking primary antibody to identify non-specific HRP activity.

• Signal variability between replicates:

  • Solution: Standardize sample preparation, antibody dilution, and incubation times.

  • Solution: Prepare fresh working dilutions of HRP-conjugated antibody for each experiment, as repeated freeze-thaw cycles can affect activity.

  • Solution: Consider ambient temperature fluctuations that may affect enzyme kinetics; maintain consistent temperature during substrate development.

How can researchers validate the specificity of NCR3 Antibody binding across different experimental conditions?

Validating antibody specificity is crucial for reliable experimental results. For NCR3 Antibody, HRP conjugated, researchers should implement multiple validation strategies:

• Positive and negative control samples:

  • Use cell lines with known NCR3 expression (e.g., NK-92 cells) as positive controls.

  • Use cell lines lacking NCR3 expression (e.g., most epithelial cell lines) or NCR3 knockout models as negative controls.

  • Compare staining patterns between these controls under identical experimental conditions.

• Peptide competition assays:

  • Pre-incubate the antibody with excess recombinant NCR3 protein (19-135AA region as specified in the product information) .

  • Compare signal between samples with and without peptide competition.

  • Specific binding should be significantly reduced or eliminated when the antibody is pre-absorbed with its target antigen.

• Orthogonal method validation:

  • Confirm NCR3 presence using alternative detection methods such as RT-PCR or mass spectrometry.

  • Compare protein expression patterns detected by antibodies targeting different epitopes of NCR3.

  • Correlation between detection methods increases confidence in antibody specificity.

• Antibody dilution series:

  • Perform a dilution series to identify the optimal antibody concentration.

  • Specific binding should show a dose-dependent pattern, while non-specific binding often shows less consistent patterns with dilution.

• Cross-species reactivity testing:

  • While the antibody is designed for human NCR3 detection , testing against samples from other species can help establish specificity boundaries.

  • Document any cross-reactivity for future reference and experimental planning.

• Denatured vs. native conditions:

  • Test antibody performance in assays requiring denatured proteins (Western blot) versus native conformation (ELISA, flow cytometry).

  • Some antibodies perform differently under these conditions, affecting experimental design choices.

Implementing these validation approaches not only confirms antibody specificity but also helps optimize experimental conditions for different applications, ultimately enhancing data reliability and reproducibility.

How can NCR3 Antibody be incorporated into strategies for enhancing NK cell-mediated immunotherapies?

The development of effective NK cell-mediated immunotherapies represents a promising frontier in cancer treatment. NCR3 Antibody can be strategically incorporated into several advanced immunotherapeutic approaches:

Research has demonstrated that high-affinity NCR3-targeting antibodies converted into bispecific formats can effectively redirect NK cytotoxicity toward tumor cells, suggesting that this receptor represents a viable target for immunotherapeutic development comparable to the more extensively studied CD16 and NCR1 .

What are the technical considerations for studying NCR3's role in dendritic cell maturation?

NCR3 plays a significant role in dendritic cell (DC) maturation through its interaction with ligands like BAG6 . Studying this process presents unique technical challenges and opportunities:

By carefully addressing these technical considerations, researchers can gain deeper insights into how NCR3-mediated interactions shape DC populations and influence the development of adaptive immune responses, potentially informing new approaches to therapeutic vaccination strategies.