Allergen Ara h 1, clone P17 Antibody, HRP conjugated

What is Allergen Ara h 1, clone P17 and why is it significant in immunological research?

Allergen Ara h 1, clone P17 is a variant of the major peanut allergen Ara h 1 found in peanuts (Arachis hypogaea). It belongs to the vicilin/7S globulin protein family and significantly contributes to peanut allergies alongside Ara h 2 and Ara h 3. The allergenicity of Ara h 1 has been specifically linked to the arrangement of monomers in its homotrimeric structure .

Ara h 1 is classified as a bicupin storage protein. The cDNA sequences of two Ara h 1 encoding clones, 41B and P17, were published in 1995, showing sequence identity greater than 97% and encoding proteins of approximately 68 kDa. Both proteins contain an N-terminal 25 amino acid residue signal peptide and a single glycosylation site (NAS) at positions 521-523 .

Its significance in immunological research stems from its role as one of the major peanut allergens responsible for IgE-mediated allergic reactions, making it an essential target for studying food allergy mechanisms and developing diagnostic methods.

How do recombinant and natural versions of Ara h 1 differ in their structural properties?

The structural differences between recombinant and natural Ara h 1 are substantial and impact experimental applications:

• Small angle x-ray scattering (SAXS) studies reveal that natural Ara h 1 (nAra h 1) forms higher molecular weight aggregates in solution.

• In contrast, full-length recombinant Ara h 1 (rAra h 1) is partially unfolded and exists as a monomer.

• The crystal structure of the Ara h 1 core region (residues 170-586) shows a bicupin fold .

• In its crystalline state, the core region forms trimeric assemblies, while in solution it exists as higher molecular weight assemblies, indicating that the core region is sufficient for trimer and oligomer formation .

These differences are crucial to consider when designing experiments, as the quaternary structure can affect epitope presentation and antibody recognition.

What are the recommended applications for Allergen Ara h 1, clone P17 Antibody, HRP conjugated?

The Allergen Ara h 1, clone P17 Antibody, HRP conjugated is primarily optimized for the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The antibody is specifically validated for ELISA applications, making it valuable for quantitative detection of Ara h 1 in research samples .

• Immunoassays: The HRP conjugation allows for direct detection without requiring secondary antibodies, simplifying workflow and potentially reducing background signal .

• Food allergen detection: The antibody can be used in detection systems for identifying peanut contamination in food products .

When using this antibody, researchers should optimize dilutions empirically for their specific applications. The recommended dilution ranges should be determined by end users based on their experimental conditions .

How can I develop a sandwich ELISA using Allergen Ara h 1, clone P17 Antibody, HRP conjugated?

Developing a sandwich ELISA using this antibody requires careful optimization of several parameters:

• Capture antibody selection: For a sandwich ELISA, you'll need a complementary antibody that recognizes a different epitope than the HRP-conjugated detection antibody. Pairwise interaction analysis can help identify optimal antibody pairs .

• Protocol outline:

  • Coat microplates with capture antibody in carbonate buffer (pH 9.6) overnight at 4°C

  • Block with BSA or similar blocking agent

  • Add samples or standards containing Ara h 1

  • Apply the HRP-conjugated Allergen Ara h 1 clone P17 antibody

  • Develop with TMB substrate and measure absorbance at 450 nm

• Optimization variables:

  • Antibody concentrations

  • Incubation times and temperatures

  • Washing steps

  • Blocking conditions

For maximum sensitivity and specificity, consider performing checkerboard titrations to determine optimal antibody concentrations and conducting spike recovery tests to validate assay performance in your specific sample matrix.

What sample preparation methods are recommended when working with this antibody for food allergen detection?

Effective sample preparation is crucial for accurate detection of Ara h 1 in food matrices:

• For protein extraction from peanuts or food samples:

  • Grind samples to fine powder

  • Extract proteins in appropriate buffer (commonly PBS with detergents)

  • Consider defatting samples to improve extraction efficiency

  • Perform clarification by centrifugation (typically at ~10,000 g for 30 min at 4°C)

• For complex food matrices:

  • Liquid-liquid extraction may be necessary before tryptic digestion to remove interfering compounds

  • For highly processed foods, consider increased extraction time or alternative buffers

• Processing for mass spectrometry analysis (if confirming ELISA results):

  • Follow extraction with reduction, alkylation, liquid-liquid extraction, and denaturation

  • Perform tryptic digestion to generate peptides for analysis

  • Note that surfactants during digestion might not improve results for all peptides

Importantly, when analyzing for potential cross-reactivity with other legumes or nuts, include appropriate controls to ensure specificity of detection.

How can I validate the specificity of the Allergen Ara h 1, clone P17 Antibody, HRP conjugated?

Validating antibody specificity is essential for reliable research results. For Allergen Ara h 1, clone P17 Antibody, implement these approaches:

• Cross-reactivity testing:

  • Test against other peanut allergens (Ara h 2, Ara h 3, etc.)

  • Evaluate reactivity with allergens from related legumes and nuts

  • Include non-allergenic proteins as negative controls

• Epitope validation:

  • Compare antibody recognition sites with known epitopes of Ara h 1

  • The clone P17 targets a specific region (amino acids 26-216), so competitive binding assays with synthetic peptides from this region can confirm specificity

• Western blot analysis:

  • Run purified Ara h 1 alongside potential cross-reactive proteins

  • Confirm binding pattern matches the expected molecular weight (~68 kDa for full-length Ara h 1)

• Mass spectrometry confirmation:

  • Use immunoprecipitation followed by MS analysis to confirm the identity of captured proteins

  • This approach can identify any non-specific interactions

Remember that this antibody has been raised against recombinant Peanut Allergen Ara h 1, clone P17 protein (amino acids 26-216) , so it may not recognize all processed forms of the protein equally.

How can I use this antibody to investigate T cell responses to Ara h 1 in peanut allergy research?

For investigating T cell responses to Ara h 1 in peanut allergy research, this antibody can be incorporated into several advanced experimental approaches:

• Combined with tetramer-guided epitope mapping (TGEM):

  • Use the antibody to isolate Ara h 1 from samples

  • Process the isolated protein for presentation to T cells

  • Utilize HLA class II/Ara h 1-specific tetramers to identify and track Ara h 1-reactive T cells

  • This approach has revealed that Ara h 1-reactive T cells are present at approximately 9 cells per million in peanut-allergic subjects compared to less than 1 cell per million in non-atopic subjects or atopic subjects without peanut allergy

• For T cell stimulation assays:

  • Capture Ara h 1 using this antibody from complex mixtures

  • Present to antigen-presenting cells (APCs)

  • Evaluate T cell proliferation and cytokine responses

• Characterizing phenotypes of Ara h 1-reactive T cells:

  • Research has shown that Ara h 1-reactive T cells express CCR4 but not CRTH2, distinguishing them from pollen-reactive T cells that express both markers

  • The percentage of Ara h 1-reactive cells expressing β7 integrin is lower compared to total CD4+ T cells

This approach allows examination of the specific T cell populations involved in peanut allergic responses, which can inform immunotherapy development.

What strategies can be employed to analyze Ara h 1 epitopes using this antibody?

The analysis of Ara h 1 epitopes requires sophisticated methodological approaches that integrate this antibody with additional techniques:

• Multi-pin overlapping peptide methods:

  • Synthesize overlapping peptides (e.g., 20-amino-acid length with 10-amino-acid overlapping) based on Ara h 1 protein sequence

  • Screen these peptides against the antibody to identify specific binding regions

  • Refine binding regions with shorter peptides to identify core epitopes

  • Previous research using this approach identified QEWGTPGS as a core linear epitope for another anti-Ara h 1 antibody

• Alanine scanning mutagenesis:

  • For identified epitopes, create peptides with alanine substitutions at each position

  • Test binding of the antibody to these substituted peptides

  • Determine which amino acids are critical for antibody recognition

  • Research has shown that substituting QEW with alanine abolished binding of a human monoclonal antibody to Ara h 1, indicating these residues are critical for recognition

• Structural analysis integration:

  • Map identified epitopes onto the known crystal structure of Ara h 1

  • Determine if epitopes are located on the surface or buried in the protein

  • Assess how oligomerization affects epitope accessibility

These approaches provide detailed information about antibody-antigen interactions and can inform both diagnostic and therapeutic developments.

How does the structural conformation of Ara h 1 affect epitope recognition by this antibody?

The structural conformation of Ara h 1 significantly impacts epitope recognition, which is crucial for experimental design and interpretation:

• Oligomerization effects:

  • Natural Ara h 1 forms higher molecular weight aggregates in solution, while full-length recombinant Ara h 1 exists as a partially unfolded monomer

  • The core region of Ara h 1 (residues 170-586) forms trimeric assemblies in the crystalline state and higher molecular weight assemblies in solution

  • These different quaternary structures can mask or expose specific epitopes

• Bicupin fold implications:

  • The central part of Ara h 1 has a bicupin fold that creates a specific three-dimensional arrangement of potential epitopes

  • The crystal structure data (PDB codes: 3s7e and 3s7i) provides detailed information about this fold:

When designing experiments, consider that the recombinant protein used to generate this antibody (amino acids 26-216) may fold differently than the same region in the native, full-length protein, potentially affecting epitope recognition.

What are common sources of background signal when using this HRP-conjugated antibody, and how can they be minimized?

Background signal can compromise assay sensitivity and specificity. When using this HRP-conjugated antibody, consider these common sources and mitigation strategies:

• Non-specific binding:

  • Increase blocking agent concentration (try 2-5% BSA or non-fat dry milk)

  • Add 0.05-0.1% Tween-20 to washing and antibody diluent buffers

  • Consider including 1-5% normal serum from the same species as your samples

• Cross-reactivity with similar allergens:

  • Pre-absorb the antibody with potential cross-reactive proteins

  • Increase washing stringency with higher salt concentrations

  • Optimize antibody dilution to minimize non-specific interactions

• Matrix effects from food samples:

  • Implement additional sample clean-up steps (e.g., solid-phase extraction)

  • Prepare standards in a matrix similar to your samples

  • Include appropriate extraction controls

• Endogenous peroxidase activity:

  • Treat samples with 0.3% H₂O₂ in methanol for 30 minutes

  • Consider using alternative detection systems if persistent

• Reagent contamination:

  • Use freshly prepared buffers and substrates

  • Store the antibody at recommended temperatures (-20°C or -80°C)

  • Avoid repeated freeze-thaw cycles

Systematic optimization through checkerboard titrations and careful selection of negative controls can help identify the specific source of background in your experimental system.

How can I improve the detection limit of assays using Allergen Ara h 1, clone P17 Antibody, HRP conjugated?

Improving detection limits requires systematic optimization of multiple parameters:

• Signal amplification strategies:

  • Consider tyramide signal amplification (TSA) to enhance the HRP signal

  • Implement longer substrate incubation times at controlled temperatures

  • Explore alternative substrates with higher sensitivity (e.g., SuperSignal ELISA Femto)

• Sample concentration techniques:

  • Use solid-phase extraction to concentrate Ara h 1 from dilute samples

  • Implement immunoprecipitation to enrich the target protein

  • Ammonium sulfate precipitation can be effective (70-100% saturation has been used for Ara h 1 isolation)

• Instrument sensitivity:

  • Optimize plate reader settings (integration time, wavelength, etc.)

  • Consider using more sensitive detection platforms like chemiluminescence readers

  • When using mass spectrometry for confirmation, high-sensitivity triple quadrupole instruments can detect as little as 2 ppm of peanut allergen Ara h 1 in complex matrices

• Assay format modifications:

  • Implement a sequential instead of simultaneous incubation in sandwich assays

  • Extend incubation times at 4°C to enhance binding equilibrium

  • Optimize washing procedures to reduce non-specific binding without removing specific signal

Documenting each optimization step systematically will help establish the most sensitive protocol for your specific application.

What considerations should be made when analyzing Ara h 1 in processed food samples?

Analyzing Ara h 1 in processed food samples presents unique challenges requiring specialized approaches:

• Protein extraction efficiency:

  • Processing can create protein-matrix interactions that reduce extraction efficiency

  • Consider using stronger extraction buffers containing detergents (SDS, Triton X-100) and reducing agents

  • Increase extraction time and include sonication steps to improve yield

  • For highly processed foods, higher temperatures during extraction may be necessary

• Epitope modifications:

  • Thermal processing and Maillard reactions can modify epitopes

  • The antibody was raised against recombinant protein (amino acids 26-216) , so it may not recognize all processed forms equally

  • Include appropriate processed peanut standards in your analysis

• Matrix interference:

  • Food matrices contain components that can interfere with antibody binding

  • Implement matrix-matched calibration curves

  • Consider including sample-specific validation using spike recovery tests

  • Liquid-liquid extraction before analysis has been shown to reduce matrix effects

• Cross-reactivity assessment:

  • Processed foods often contain multiple ingredients that may cross-react

  • Include negative controls containing potential cross-reactive ingredients without peanut

  • Consider confirmatory testing with orthogonal methods such as mass spectrometry

• Protein fragmentation:

  • Processing can fragment proteins, potentially separating epitopes

  • The antibody targets a specific region (26-216AA) , so fragmentation may affect detection

  • Consider using antibody combinations targeting different regions in sandwich formats

These considerations are especially important for food safety applications, where accurate detection of allergens is critical.

How might this antibody be used to study the relationship between Ara h 1 structure and allergenicity?

This antibody can serve as a valuable tool for investigating the complex relationship between Ara h 1 structure and allergenicity:

• Conformational studies:

  • Use the antibody to detect structural changes in Ara h 1 under different conditions

  • Compare binding to natural versus recombinant forms, which differ significantly in their structural properties

  • Assess how processing conditions affect antibody recognition and correlate with allergenicity

• Epitope mapping in relation to quaternary structure:

  • Since natural Ara h 1 forms higher-order oligomers while recombinant versions may not , the antibody can help identify epitopes that are exposed or hidden in different oligomeric states

  • This information can provide insight into why certain structural conformations are more allergenic

• Integration with T cell studies:

  • Combine antibody-based isolation of specific Ara h 1 conformations with T cell response assays

  • Research has shown that Ara h 1-reactive T cells in allergic subjects express specific surface markers like CCR4 but not CRTH2

  • This approach can help determine which conformational states are most effective at triggering allergic responses

• Stability and digestion resistance analysis:

  • Use the antibody to track Ara h 1 during simulated digestion processes

  • Correlate structural stability with allergenicity

  • Ara h 1 is known to be resistant to proteases, heat, and denaturants , and this antibody can help monitor how these properties relate to allergenic potential

These studies can provide critical insights for developing hypoallergenic variants or more effective immunotherapies.

What role could this antibody play in developing novel immunotherapeutic approaches for peanut allergy?

This antibody could significantly contribute to developing novel immunotherapeutic approaches through several research avenues:

• Identifying immunodominant epitopes:

  • Use competitive binding assays with patient sera to identify epitopes recognized by both this antibody and IgE from allergic individuals

  • Map these epitopes to the known structure of Ara h 1 to inform therapeutic design

  • Previous research has identified multiple T cell epitopes in Ara h 1 with defined HLA restriction

• Monitoring modified allergens:

  • Track structural changes in allergens designed for immunotherapy

  • Verify that modified allergens retain critical epitopes for desensitization while reducing IgE binding

  • Compare binding patterns to natural and recombinant forms of Ara h 1

• Developing bifunctional molecules:

  • Create conjugates of this antibody with immunomodulatory molecules

  • Target allergen-specific regulatory mechanisms

  • Monitor the efficiency of such constructs in redirecting immune responses

• Evaluating response to immunotherapy:

  • Use the antibody to develop assays that track changes in Ara h 1-specific antibody profiles during treatment

  • Monitor shifts from IgE to IgG4 responses against the same epitopes

  • Correlate these changes with clinical outcomes

• Investigating adjuvant formulations:

  • Assess how different adjuvants affect the presentation and processing of Ara h 1

  • Use the antibody to track allergen release kinetics from adjuvant formulations

  • Optimize delivery systems for immunotherapy

The combination of structural insights and epitope mapping facilitated by this antibody could lead to more targeted and effective immunotherapeutic approaches.

How can this antibody contribute to understanding cross-reactivity between peanut and other legume allergens?

This antibody provides a valuable tool for investigating the molecular basis of cross-reactivity between peanut and other legume allergens:

• Comparative epitope analysis:

  • Use the antibody in competitive binding assays with potential cross-reactive proteins

  • Determine if shared epitopes exist between Ara h 1 and homologous proteins from other legumes

  • Synthesize peptides from homologous regions of related proteins to precisely map cross-reactive epitopes

• Structural homology assessment:

  • Apply bioinformatics approaches to compare the region recognized by this antibody (amino acids 26-216) with similar regions in other legume proteins

  • Previous work has used CLANS clustering of Ara h 1 homologous sequences based on pairwise BLAST similarity scores with a p-value threshold of 10^-3 to identify related proteins

  • Experimental validation of predicted cross-reactivity can be performed using this antibody

• Protein modification effects:

  • Investigate how post-translational modifications affect cross-reactivity

  • Ara h 1 has a single glycosylation site (NAS) at positions 521-523

  • Evaluate if similar modifications occur in homologous proteins and how they impact antibody recognition

• Clinical correlation studies:

  • Use the antibody in inhibition assays with sera from patients with multiple legume allergies

  • Correlate molecular cross-reactivity detected by the antibody with clinical cross-reactivity

  • Develop predictive models for clinical cross-reactivity based on epitope conservation

This research direction has significant clinical implications, as understanding cross-reactivity mechanisms could improve risk assessment and management for individuals with peanut allergies who may react to other legumes.