BB3856 Antibody

What is BB3856 Antibody and what is its target protein?

BB3856 Antibody is a polyclonal antibody raised in rabbits that specifically targets Azurin (BB3856) from Bordetella bronchiseptica. The target protein has the UniProt accession number P0A321 and is classified as an azurin protein, which is a small copper-containing protein involved in electron transfer processes. This antibody has been generated using recombinant Bordetella bronchiseptica BB3856 protein as the immunogen, specifically from the strain ATCC BAA-588 / NCTC 13252 / RB50 (formerly known as Alcaligenes bronchisepticus) .

The antibody recognizes the native conformational structure of the target protein and is suitable for various research applications including ELISA and Western Blotting techniques. It's important to note that this antibody is intended exclusively for research purposes and not for diagnostic or therapeutic applications .

What validated applications exist for BB3856 Antibody in research settings?

Based on available technical documentation, BB3856 Antibody has been validated for the following experimental applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): This application allows for the quantitative detection of the target protein in solution or bound to solid phases.

• Western Blotting (WB): The antibody has been validated for identifying the target protein following SDS-PAGE separation and membrane transfer.

Both applications ensure proper identification of the target antigen in experimental settings. The antibody's polyclonal nature means it recognizes multiple epitopes on the target protein, potentially providing stronger signal detection compared to monoclonal antibodies that recognize single epitopes .

What are the recommended storage and handling conditions for BB3856 Antibody?

For optimal performance and stability of BB3856 Antibody, the following storage and handling protocols are recommended:

• Upon receipt, store the antibody at -20°C or -80°C for long-term preservation.

• Avoid repeated freeze-thaw cycles as these can compromise antibody integrity and function.

• The antibody is supplied in liquid form with a storage buffer composition consisting of:

  • 0.03% Proclin 300 (preservative)

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

These components help maintain antibody stability during storage. When handling the antibody, aseptic techniques should be employed to prevent contamination. Prior to use, it's advisable to gently mix the antibody solution by inversion rather than vortexing, which may cause protein denaturation .

How can researchers optimize experimental protocols for BB3856 Antibody in Western Blotting?

Optimizing Western Blotting protocols for BB3856 Antibody requires systematic approach to several key parameters:

Sample Preparation:

• When extracting proteins from B. bronchiseptica cultures, use lysis buffers containing protease inhibitors to prevent degradation of the target protein.

• Optimal protein loading typically ranges from 20-50 μg of total protein per lane, depending on expression levels of the target.

• For bacterial samples, sonication followed by centrifugation at 14,000g for 15 minutes at 4°C is often effective for protein extraction.

Blocking and Antibody Incubation:

• Start with a 1:1000 dilution of the antibody in blocking buffer (5% non-fat dry milk or BSA in TBST).

• Incubate membranes with primary antibody solution overnight at 4°C with gentle rocking.

• Perform stringent washing steps (3-5 washes of 5-10 minutes each) with TBST to reduce background.

Detection Optimization:

• Use HRP-conjugated anti-rabbit secondary antibodies at 1:5000 dilution.

• For enhanced sensitivity, consider using chemiluminescent substrates with extended signal duration.

• If signal-to-noise ratio is low, try adjusting antibody concentration or implementing more stringent washing protocols.

When troubleshooting unexpected results, consider that the polyclonal nature of this antibody may result in detection of multiple bands if the target protein undergoes post-translational modifications or exists in multiple isoforms .

What are the key considerations for designing ELISA protocols with BB3856 Antibody?

When designing ELISA protocols using BB3856 Antibody, researchers should consider the following technical aspects:

Plate Coating:

• For direct ELISA, coat plates with purified Azurin protein (1-10 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C.

• For sandwich ELISA, use a capture antibody against a different epitope of Azurin before applying your sample.

Antibody Titration:

• Perform an initial antibody titration (typically starting from 1:500 to 1:10,000) to determine optimal concentration.

• Plot signal-to-noise ratios against antibody concentrations to identify the dilution that maximizes specific signal while minimizing background.

Assay Validation:

• Include positive controls (purified Azurin protein) and negative controls (unrelated bacterial proteins).

• Establish a standard curve using purified recombinant Azurin at concentrations ranging from 0.1-1000 ng/ml.

• Calculate assay sensitivity and dynamic range based on this standard curve.

Cross-Reactivity Testing:

• Evaluate potential cross-reactivity with azurin proteins from related Bordetella species or other bacteria expressing similar copper-binding proteins.

• Document any observed cross-reactivity to ensure accurate interpretation of experimental results.

Implementation of these methodological considerations will help ensure reliable and reproducible ELISA results when using BB3856 Antibody for quantitative detection of Azurin .

How does BB3856 Antibody perform in immunohistochemistry applications?

While the product documentation does not specifically validate BB3856 Antibody for immunohistochemistry (IHC) applications, researchers interested in exploring this application should consider the following experimental approach:

Protocol Development:

• Begin with standard IHC protocols using formalin-fixed, paraffin-embedded (FFPE) sections of B. bronchiseptica-infected tissues.

• Test multiple antigen retrieval methods, starting with heat-induced epitope retrieval using citrate buffer (pH 6.0) and EDTA buffer (pH 9.0).

• Implement a titration series (1:100 to 1:1000) to determine optimal antibody concentration.

Controls and Validation:

• Include positive control tissues (confirmed B. bronchiseptica-infected samples) and negative controls (uninfected tissues and primary antibody omission).

• Consider parallel validation with alternative detection methods (PCR, bacterial culture) to confirm specificity.

Optimization Strategies:

• If signal strength is insufficient, consider signal amplification systems such as tyramide signal amplification (TSA).

• For high background, implement more stringent blocking (using 10% normal serum from the same species as the secondary antibody) and extended washing steps.

It's important to note that any IHC application would require extensive validation by individual laboratories, as this is not a vendor-validated application for this particular antibody. When reporting results, researchers should thoroughly document all optimization steps and validation controls .

How can BB3856 Antibody be incorporated into Fc-dependent functional studies?

Incorporating BB3856 Antibody into Fc-dependent functional studies requires understanding of both antibody mechanisms and experimental design considerations:

Experimental Design Principles:

• For antibody-dependent cellular cytotoxicity (ADCC) assays, researchers can use BB3856 Antibody to target Bordetella bronchiseptica-infected cells that express surface-exposed Azurin.

• For antibody-dependent cell-mediated phagocytosis (ADCP) studies, macrophages or neutrophils can be co-cultured with opsonized bacteria or infected cells.

• Comparative studies with different antibody isotypes can help elucidate the role of Fc-mediated functions in host defense against B. bronchiseptica.

Methodological Approach:

• Prepare target cells expressing BB3856 Azurin protein (either through infection or transfection).

• Pre-incubate target cells with BB3856 Antibody at concentrations ranging from 0.1-10 μg/ml.

• Add effector cells (NK cells for ADCC; macrophages for ADCP) at various effector-to-target ratios (typically 5:1 to 50:1).

• Measure cytotoxicity using standard assays (LDH release, 51Cr release) or phagocytosis via flow cytometry with fluorescently-labeled targets.

It's important to note that understanding Fc-dependent mechanisms of antibody function represents a significant area of current immunological research, as highlighted by the NIH funding opportunity (RFA-AI-23-054) focused on elucidating these pathways. While this specific research direction has not been explicitly validated for BB3856 Antibody, the polyclonal nature of this rabbit IgG antibody makes it potentially suitable for such investigations .

What are the key considerations when using BB3856 Antibody for studying Bordetella bronchiseptica virulence factors?

When using BB3856 Antibody to study Azurin as a potential virulence factor in Bordetella bronchiseptica, researchers should consider the following experimental design elements:

Experimental Models:

• In vitro infection models using relevant cell lines (e.g., respiratory epithelial cells, macrophages).

• Animal models of B. bronchiseptica infection, particularly in the respiratory tract.

• Comparative studies between wild-type bacteria and Azurin-deficient mutants.

Technical Approaches:

• Immunofluorescence microscopy to visualize Azurin localization during infection using BB3856 Antibody.

• Immunoprecipitation to identify potential host-pathogen protein-protein interactions involving Azurin.

• ELISA-based quantification of Azurin expression under various environmental conditions.

Biological Questions to Address:

• Is Azurin secreted during infection, and if so, does it modulate host immune responses?

• Does Azurin expression correlate with bacterial survival in different host microenvironments?

• Can neutralization of Azurin using BB3856 Antibody alter the course of infection?

Controls and Validations:

• Include isotype control antibodies to account for non-specific effects.

• Validate findings using genetic approaches (gene knockout, complementation).

• Consider potential cross-reactivity with host proteins containing copper-binding domains.

By integrating these considerations into experimental design, researchers can effectively utilize BB3856 Antibody to advance understanding of Bordetella pathogenesis and host-pathogen interactions .

How does BB3856 Antibody compare with other antibodies targeting bacterial copper-binding proteins?

When comparing BB3856 Antibody to other antibodies targeting bacterial copper-binding proteins, researchers should consider several comparative parameters:

Specificity Profile:

• BB3856 Antibody specifically targets Azurin from Bordetella bronchiseptica (UniProt P0A321).

• Other copper-binding protein antibodies may target azurins from different bacterial species (e.g., Pseudomonas aeruginosa), plastocyanins, or other copper-containing bacterial proteins.

• Cross-reactivity testing is essential when working with closely related bacterial species.

Experimental Applications Comparison:

Functional Considerations:

• Azurins from different bacterial species share structural similarities but may have species-specific functional roles.

• When designing comparative studies, consider that different copper-binding proteins may have distinct subcellular localizations and expression patterns.

• For evolutionary studies, select antibodies that can distinguish between closely related protein homologs.

When planning experiments requiring multiple antibodies against bacterial copper-binding proteins, consider the compatibility of detection systems, potential cross-reactivity issues, and whether polyclonal or monoclonal antibodies are more appropriate for your specific research questions .

What are the most common causes of false positive or false negative results when using BB3856 Antibody?

Understanding potential sources of error when using BB3856 Antibody can help researchers troubleshoot experimental issues:

Potential Causes of False Positive Results:

• Cross-reactivity with structurally similar proteins, particularly other copper-binding proteins in the sample.

• Insufficient blocking leading to non-specific binding of the antibody to the membrane or plate.

• Overly sensitive detection systems that amplify background signal.

• Contamination of samples with actual target protein from other sources.

Mitigation Strategies for False Positives:

• Implement more stringent washing procedures (increase number and duration of washes).

• Optimize blocking conditions (test different blocking agents and concentrations).

• Perform pre-adsorption of the antibody with non-specific proteins.

• Include appropriate negative controls lacking the target protein.

Potential Causes of False Negative Results:

• Target protein denaturation during sample preparation, destroying the epitopes recognized by the antibody.

• Insufficient antigen concentration in the sample.

• Interference from sample components that mask epitopes or inhibit antibody binding.

• Degradation of the antibody due to improper storage or handling.

Mitigation Strategies for False Negatives:

• Optimize protein extraction conditions to preserve native protein structure.

• Concentrate samples if target protein is present at low levels.

• Test alternative sample preparation methods to reduce interference.

• Verify antibody activity using positive control samples containing known quantities of target protein.

Implementing these troubleshooting approaches can significantly improve reliability when using BB3856 Antibody in research applications .

How can researchers validate BB3856 Antibody for novel applications beyond the manufacturer's recommendations?

Validating BB3856 Antibody for novel applications requires a systematic approach to ensure reliable and reproducible results:

Step-by-Step Validation Process:

• Positive Control Testing:

  • Use purified recombinant Azurin protein as a positive control.

  • Demonstrate specific binding in the established applications (ELISA, WB) first.

  • Document the expected signal pattern before expanding to new applications.

• Specificity Confirmation:

  • Perform parallel experiments with pre-immune serum or isotype control antibodies.

  • Consider using competitive inhibition with excess antigen to confirm specific binding.

  • For genetic confirmation, compare wild-type B. bronchiseptica with azurin-deficient mutants.

• Titration and Optimization:

  • For each new application, conduct a systematic titration series.

  • Optimize all protocol parameters (buffer composition, incubation times, detection methods).

  • Document optimization steps methodically for reproducibility.

• Cross-Platform Validation:

  • Confirm findings using orthogonal detection methods.

  • For example, validate immunofluorescence results with Western blotting.

  • Consider mass spectrometry-based verification of immunoprecipitated proteins.

Example Validation Framework for Immunoprecipitation:

• Start with lysate from B. bronchiseptica expressing high levels of Azurin.

• Compare BB3856 Antibody with control IgG at equivalent concentrations.

• Analyze precipitated proteins by Western blot using a different anti-Azurin antibody (if available).

• Confirm identity of precipitated proteins by mass spectrometry.

• Document antibody amount, bead type, washing conditions, and elution method.

This systematic approach to validation ensures that any novel application of BB3856 Antibody is scientifically rigorous and reproducible across different experimental settings .

What considerations should be made when using BB3856 Antibody in multiplex immunoassays?

Integrating BB3856 Antibody into multiplex immunoassay systems requires careful consideration of several technical factors:

Antibody Compatibility Factors:

• Cross-Reactivity Assessment:

  • Test BB3856 Antibody against all proteins included in the multiplex panel.

  • Document any observed cross-reactivity to ensure accurate data interpretation.

  • Consider pre-absorption procedures if cross-reactivity is detected.

• Signal Interference Evaluation:

  • Assess potential fluorescence spillover when using fluorophore-conjugated antibodies.

  • Determine if the signal from BB3856 Antibody detection interferes with other assay components.

  • Implement appropriate compensation controls if using flow cytometry-based multiplex systems.

• Dynamic Range Considerations:

  • Ensure that the detection range for Azurin is compatible with other analytes in the multiplex.

  • Adjust antibody concentrations to achieve comparable signal intensities across all targets.

  • Verify that high-abundance targets don't mask detection of low-abundance proteins.

Optimization Strategies:

• Sequential Detection Approach:

  • Consider sequential rather than simultaneous detection if interference is observed.

  • Determine optimal order of antibody application to minimize cross-reactivity.

• Antibody Labeling Considerations:

  • If direct labeling of BB3856 Antibody is required, select fluorophores or enzymes that provide optimal separation from other detection channels.

  • Validate that labeling procedures don't compromise antibody specificity or sensitivity.

• Data Analysis Adjustments:

  • Implement appropriate statistical methods to account for background and cross-reactivity.

  • Consider standard curve adjustments when analyzing multiplex data.

By addressing these considerations systematically, researchers can successfully incorporate BB3856 Antibody into multiplex immunoassays for simultaneous detection of multiple bacterial proteins or host response markers .

How might BB3856 Antibody contribute to research on bacterial copper homeostasis and virulence?

BB3856 Antibody targeting Azurin presents several promising avenues for investigating the relationship between copper homeostasis and bacterial virulence:

Research Opportunities:

• Copper Trafficking Studies:

  • Using BB3856 Antibody to track Azurin localization during different growth phases and stress conditions.

  • Investigating how copper limitation or excess affects Azurin expression and localization.

  • Exploring potential interactions between Azurin and host copper-binding proteins during infection.

• Virulence Mechanism Investigation:

  • Determining whether Azurin contributes to B. bronchiseptica survival in copper-restricted host environments.

  • Exploring potential roles of Azurin in oxidative stress resistance during host-pathogen interactions.

  • Investigating whether Azurin functions in redox signaling pathways that regulate virulence gene expression.

• Host-Pathogen Interaction Studies:

  • Using BB3856 Antibody to visualize Azurin during different stages of infection.

  • Determining whether host immune cells recognize and respond to bacterial Azurin.

  • Investigating if neutralization of Azurin with BB3856 Antibody alters infection outcomes.

Experimental Approaches:

• Combine BB3856 Antibody-based detection with genetic manipulation of copper transport systems.

• Implement transcriptomic and proteomic analyses to identify genes co-regulated with Azurin.

• Develop in vivo imaging techniques using labeled BB3856 Antibody to track Azurin during infection.

The integration of BB3856 Antibody into these research frameworks could significantly advance understanding of how bacterial copper metabolism contributes to pathogenesis and potentially reveal new therapeutic targets for combating Bordetella infections .

What potential exists for using BB3856 Antibody in developing advanced diagnostic tools for Bordetella infections?

While BB3856 Antibody is currently designated for research use only, its specificity for Bordetella bronchiseptica Azurin suggests potential applications in advanced diagnostic development:

Diagnostic Research Directions:

• Multiplex Pathogen Detection Systems:

  • Integration of BB3856 Antibody into antibody arrays for simultaneous detection of multiple respiratory pathogens.

  • Development of lateral flow assays targeting bacterial protein biomarkers rather than conventional nucleic acid detection.

  • Creation of biosensor platforms that utilize immobilized BB3856 Antibody for rapid Bordetella detection.

• Sample Type Optimization:

  • Validation of BB3856 Antibody performance across diverse clinical sample types (nasopharyngeal swabs, sputum, bronchoalveolar lavage).

  • Determination of minimum bacterial load required for reliable detection in each sample type.

  • Development of sample processing protocols to maximize Azurin recovery and detection.

• Comparative Diagnostic Studies:

  • Evaluation of Azurin detection versus conventional diagnostic methods (culture, PCR) for sensitivity and specificity.

  • Assessment of Azurin as a biomarker for different stages of infection or colonization.

  • Investigation of strain-specific variations in Azurin expression that might affect diagnostic reliability.

Technical Implementation Considerations:

• Signal amplification strategies to enhance detection sensitivity in direct clinical samples.

• Development of recombinant Azurin standards for assay calibration and quality control.

• Cross-reactivity assessment with related Bordetella species and other respiratory pathogens.

When pursuing these research directions, it remains essential to acknowledge that transitioning from research applications to diagnostic use would require extensive validation studies and regulatory approval processes .

How could BB3856 Antibody be applied in conjunction with emerging antibody engineering technologies?

The integration of BB3856 Antibody with advanced antibody engineering platforms offers several innovative research possibilities:

Emerging Technology Integration:

• Antibody Fragment Development:

  • Generation of Fab or scFv fragments from BB3856 Antibody for applications requiring smaller binding molecules.

  • Evaluation of fragment binding kinetics compared to the original antibody.

  • Development of bispecific antibody fragments combining Azurin recognition with binding to other bacterial targets.

• Application with IgDesign and Similar Platforms:

  • Utilizing the epitope specificity information from BB3856 Antibody to inform computational antibody design approaches.

  • Applying deep learning methods like IgDesign to develop improved anti-Azurin antibodies with enhanced specificity or affinity.

  • Designing antibody variants with optimized complementarity-determining regions (CDRs) while maintaining the essential binding characteristics.

• Therapeutic Antibody Research:

  • Exploring potential for humanized variants of BB3856 Antibody for research models.

  • Investigating Fc engineering to enhance specific effector functions (ADCC, ADCP) against Bordetella.

  • Developing antibody-antibiotic conjugates targeting Azurin-expressing bacteria.

Experimental Design Considerations:

• Comparative binding studies between original BB3856 Antibody and engineered variants.

• Functional assessment of engineered antibodies in relevant infection models.

• Epitope mapping to determine precise binding sites and inform rational antibody design.

These advanced applications align with current trends in therapeutic antibody development, as highlighted by recent breakthroughs in antibody engineering for infectious disease applications, including the Stanford-led research on SARS-CoV-2 antibodies that utilize paired antibody approaches for enhanced neutralization capacity .