Allergen Ara h 1, clone P17 Antibody

What is the molecular structure of Ara h 1, clone P17 and how does it differ from other variants?

Ara h 1, clone P17 is a bicupin storage protein of the vicilin type with a molecular weight of approximately 68 kDa. The cDNA sequence of clone P17 shows greater than 97% identity with another clone (41B) . Both encode proteins with an N-terminal 25 amino acid residue signal peptide and a single glycosylation site (NAS) at amino acid positions 521–523.

The mature protein forms after the cleavage of both the signal peptide and an N-terminal propeptide, with approximately 78-84 residues removed during post-translational processing . In SDS-PAGE, Ara h 1 isoforms appear as two closely spaced bands at 69 and 66 kDa, consistent with this processing pathway .

Structural comparison between Ara h 1 variants:

The truncated 54 kDa variant represents a new isoform with distinct physicochemical properties, suggesting functional differences between these variants in their natural context .

How is Ara h 1, clone P17 processed post-translationally and what impact does this have on its allergenicity?

Ara h 1 undergoes complex post-translational processing that significantly affects its immunogenicity:

• Pre-pro-protein translation: Initially synthesized with a 25-residue signal peptide

• Vacuolar targeting: Signal peptide directs nascent protein to storage vacuole

• Propeptide cleavage: N-terminal propeptide (53-59 residues) removed to yield mature Ara h 1

• N-glycosylation: Addition of glycans at position 521-523 (NAS motif)

• Oligomerization: Formation of stable trimers through non-covalent interactions

The N-terminal propeptide contains three allergenic epitopes, of which two are major contributors to allergenicity . Interestingly, this peptide contains six cysteine residues that might stabilize its structure against digestive denaturation and resembles a class of antifungal oligopeptides from plant seeds .

Glycoform analyses have revealed both high-mannose type and β-xylosylated type N-glycans linked to Ara h 1 . In some patients, these N-glycans serve as the sole epitope for Ara h 1-specific IgE, as demonstrated by complete loss of cross-reactivity following de-N-glycosylation .

What epitopes have been identified in Ara h 1, clone P17 and how are they characterized?

Multiple B-cell and T-cell epitopes have been identified in Ara h 1, contributing to its potent allergenicity:

B-cell (IgE) epitopes:
Five major IgE-binding epitopes have been identified that bound peanut-specific serum IgE from more than 60% of allergic patients tested . Mutational analysis of the immunodominant epitope demonstrated that single amino acid changes had dramatic effects on IgE-binding characteristics, highlighting the specificity of these interactions .

T-cell epitopes:
T-cell epitopes restricted by eight HLA class II alleles have been identified using Tetramer Guided Epitope Mapping (TGEM) . These epitopes are critical for understanding the cellular immune response to Ara h 1.

Epitope characterization methods:

• Sequential overlapping peptides: Synthetic peptides representing the Ara h 1 sequence are screened with patient sera to identify IgE-binding regions

• Tetramer Guided Epitope Mapping (TGEM): Uses HLA class II/Ara h 1-specific tetramers to identify T cell epitopes

• Mutational analysis: Single amino acid substitutions to determine critical residues for antibody binding

• Competitive inhibition assays: Synthetic peptides spanning antibody-binding sites competitively inhibit antibody reactivity, confirming epitope locations

How can Ara h 1, clone P17 antibodies be used to detect and quantify the allergen in experimental systems?

Ara h 1, clone P17 antibodies provide versatile tools for allergen detection and quantification across multiple experimental platforms:

Western blotting:
Polyclonal and monoclonal antibodies against Ara h 1, clone P17 can detect the allergen in protein extracts, with characteristic bands at 69 and 66 kDa . For enhanced detection, HRP-conjugated antibodies eliminate secondary antibody steps .

ELISA systems:

• Direct ELISA: Recombinant Ara h 1 coated on plates, detected with Ara h 1-specific antibodies

• Sandwich ELISA: Capture antibody immobilized on plate, sample added, then detection antibody

• Competitive ELISA: Sample Ara h 1 competes with plate-bound Ara h 1 for antibody binding

• Epitope-based ELISA: Synthetic peptides representing specific epitopes used as coating antigens

Immunofluorescence:
Ara h 1 antibodies can visualize the allergen in cellular contexts, providing information about localization and processing .

Protocol optimization considerations:

• Antibody dilution (typically 1:500-1:5000 for Western blot, 1:1000-1:10000 for ELISA)

• Blocking agents (BSA vs. milk proteins - avoid milk for food allergen detection)

• Detection systems (colorimetric, chemiluminescent, fluorescent)

• Sample preparation (native vs. denatured conditions)

What is the difference between using polyclonal versus monoclonal antibodies against Ara h 1, clone P17 in research applications?

The choice between polyclonal and monoclonal antibodies against Ara h 1, clone P17 significantly impacts experimental outcomes:

Polyclonal antibodies:

• Recognition: Bind multiple epitopes on Ara h 1, clone P17

• Sensitivity: Generally higher sensitivity due to multiple epitope binding

• Cross-reactivity: May cross-react with related allergens from other legumes

• Batch variability: Higher lot-to-lot variation

• Applications: Excellent for detection in complex matrices, immunoprecipitation

• Commercial availability: Available as HRP-conjugated form (>95% purity via Protein G purification)

Monoclonal antibodies:

• Recognition: Bind specific, defined epitopes on Ara h 1, clone P17

• Specificity: Higher specificity for particular Ara h 1 isoforms

• Reproducibility: Minimal lot-to-lot variation

• Epitope mapping: Valuable for precise epitope identification

• Applications: Preferred for standardized assays and epitope-specific studies

Experimental design considerations:
When designing experiments, researchers should select antibody type based on their specific goals. For quantitative detection of all Ara h 1 variants, polyclonal antibodies may offer advantages. For investigations focusing on specific epitopes or isoforms, monoclonal antibodies provide greater precision and reproducibility.

How can Ara h 1, clone P17 antibodies be used in T-cell epitope mapping studies?

Ara h 1, clone P17 antibodies serve as valuable tools in T-cell epitope mapping through multiple methodological approaches:

Tetramer Guided Epitope Mapping (TGEM):
This sophisticated technique identifies CD4+ T cell epitopes using HLA class II/Ara h 1-specific tetramers . Ara h 1 antibodies play critical roles in:

• Purifying native Ara h 1 for peptide library generation

• Verifying the identity of synthesized peptides

• Confirming the presence of Ara h 1 in antigen-presenting cell preparations

Methodology workflow:

• Generate overlapping peptides spanning the Ara h 1 sequence

• Load peptides onto antigen-presenting cells expressing specific HLA alleles

• Create HLA-peptide tetramers for peptide pools showing T cell reactivity

• Use tetramers to stain and identify epitope-specific T cells

• Confirm epitope identity with antibody blocking experiments

In a representative study, this approach identified multiple Ara h 1 epitopes with defined HLA restriction in peanut-allergic subjects, with an average frequency of Ara h 1-reactive T cells at approximately 9 cells per million in allergic individuals compared to less than 1 cell per million in non-allergic controls .

What methodologies can characterize the interaction between Ara h 1, clone P17 and the immune system?

Multiple sophisticated methodologies can characterize Ara h 1-immune system interactions at molecular and cellular levels:

T cell characterization techniques:

• Ex vivo tetramer analysis: HLA-peptide tetramers combined with magnetic bead enrichment detect rare Ara h 1-specific T cells in peripheral blood without in vitro expansion

• Multiparameter flow cytometry: Phenotyping of Ara h 1-reactive T cells for memory markers (CD45RA/RO), Th2 markers (CRTh2, CCR4), and homing markers (CLA, β7 integrin)

• Cytokine profiling: Intracellular cytokine staining or multiplex assays to detect IFN-γ, IL-4, IL-5, IL-10, and IL-17 production by Ara h 1-specific T cells

B cell and antibody analysis:

• Epitope mapping: Using sequential overlapping hexapeptides to define epitopes recognized by antibodies

• Competitive inhibition assays: Synthetic peptides competitively inhibit antibody reactivity, confirming epitope locations

• Affinity measurements: Surface plasmon resonance to determine binding kinetics between Ara h 1 and specific antibodies

Research findings:
Studies have revealed that Ara h 1-reactive T cells in allergic subjects express CCR4 but not CRTH2, with a notably low percentage expressing the gut-homing β7 integrin marker compared to total CD4+ T cells . Ara h 1-reactive cells secreting multiple cytokines (IFN-γ, IL-4, IL-5, IL-10, and IL-17) have been detected , suggesting heterogeneity in the T cell response.

What are the challenges in using recombinant Ara h 1, clone P17 for immunological studies and how can they be addressed?

Using recombinant Ara h 1, clone P17 for immunological studies presents several challenges requiring careful experimental design:

Challenge 1: Post-translational modifications

• Issue: Recombinant expression systems may not replicate native glycosylation patterns or propeptide processing

• Solution: Compare multiple expression systems (E. coli, yeast, baculovirus, mammalian cells); verify modifications by mass spectrometry; use plant-based expression systems for more authentic modifications

Challenge 2: Oligomeric state

• Issue: Natural Ara h 1 forms stable trimers and larger oligomers that impact epitope presentation

• Solution: Analyze quaternary structure by size-exclusion chromatography, analytical ultracentrifugation, or native PAGE; optimize buffer conditions to maintain native oligomeric states

Challenge 3: Purity and stability

• Issue: Contaminating proteins or degradation products can confound immunological readouts

• Solution: Employ multiple purification steps (e.g., affinity chromatography followed by size exclusion); validate purity by SDS-PAGE (>90%) ; analyze stability under experimental conditions

Challenge 4: Tag interference

• Issue: N-terminal or C-terminal tags may affect protein folding or epitope accessibility

• Solution: Compare tagged and tag-cleaved versions; position tags away from known epitopes; use small tags or no tags when possible

Experimental design recommendations:

• Include native Ara h 1 purified from peanuts as control in immunological assays

• Verify immunological equivalence between recombinant and native forms using patient sera

• Characterize recombinant proteins thoroughly before immunological testing

• Consider using the 26-614aa fragment of Ara h 1, clone P17 with N-terminal 6xHis-tag as described in commercial preparations

How do oligomerization states impact the immunogenicity and allergenicity of Ara h 1, clone P17?

The oligomerization state of Ara h 1 significantly influences its immunogenic and allergenic properties through multiple mechanisms:

Structural basis of oligomerization:
Full-length (63 kDa) Ara h 1 forms higher-order homo-oligomeric assemblies (decamers or nonamers), while truncated (54 kDa) Ara h 1 lacking the N-terminal domain occurs exclusively as a homotrimer . This indicates the N-terminal domain plays a crucial role in higher-order oligomerization.

Impact on allergenicity:

• Epitope clustering: Oligomerization creates repeating epitope arrays that can more efficiently cross-link FcεRI-bound IgE on mast cells and basophils

• Conformational epitopes: Inter-subunit interfaces may generate novel conformational epitopes absent in monomeric forms

• Protease resistance: Complex quaternary structures may shield proteolytic cleavage sites, enhancing stability during digestion

Experimental evidence:
The allergenicity of Ara h 1 has been specifically linked to the arrangement of monomers in the homotrimeric structure of vicilin/7S globulin proteins . Both the C-terminal and N-terminal domains of Ara h 1 contribute significantly to epitope formation , suggesting that different oligomeric forms may present distinct epitope repertoires.

Methodological implications:
Researchers should carefully consider oligomerization states when:

• Designing recombinant allergen constructs

• Interpreting immunological data from different Ara h 1 preparations

• Developing diagnostic reagents or immunotherapeutic approaches

• Comparing results across studies using different Ara h 1 preparations

What are the optimal storage and handling conditions for Ara h 1, clone P17 antibodies to maintain functionality?

Proper storage and handling of Ara h 1, clone P17 antibodies is critical for maintaining their specificity and activity:

Storage recommendations:

• Store at -20°C or -80°C for extended storage periods

• Avoid repeated freeze-thaw cycles that can lead to antibody degradation

• For HRP-conjugated antibodies, aliquot in single-use volumes before freezing

• Working stocks can be maintained at 4°C for up to one month

Buffer composition:
Commercial Ara h 1, clone P17 antibodies are typically supplied in:

• 50% Glycerol (cryoprotectant)

• 0.01M PBS, pH 7.4 (physiological buffer)

• 0.03% Proclin 300 (preservative)

Stability considerations:

• HRP-conjugated antibodies are more sensitive to temperature fluctuations and oxidative damage

• Polyclonal preparations may show greater stability compared to monoclonal antibodies

• Addition of carrier proteins (BSA) can enhance stability during dilution

Handling precautions:

• Follow proper thawing protocols (gradual thawing at 4°C)

• Use appropriate dilution buffers (typically PBS with 0.1-0.5% BSA)

• Avoid contamination by using sterile technique

• Protect HRP-conjugated antibodies from strong light exposure

How can researchers validate the specificity of Ara h 1, clone P17 antibodies for their specific applications?

Rigorous validation of Ara h 1, clone P17 antibodies is essential to ensure experimental reliability:

Positive controls:

• Purified native Ara h 1 from peanut extract

• Recombinant Ara h 1, clone P17 protein (26-614aa)

• Ara h 1-expressing transfected cell lines

Negative controls:

• Extracts from non-legume plant sources

• Samples processed through immunodepletion with Ara h 1-specific antibodies

• Blocking peptide competition assays

Cross-reactivity assessment:
Test antibody reactivity against:

• Other peanut allergens (Ara h 2-17)

• Homologous proteins from related legumes

• Recombinant Ara h 1 variants (truncations, mutations)

Specificity validation methods:

• Western blot: Verify single bands at expected molecular weights (69 and 66 kDa for native Ara h 1)

• Immunoprecipitation: Confirm pull-down of Ara h 1 verified by mass spectrometry

• Peptide competition: Pre-incubate antibody with synthetic peptides spanning epitope regions

• Knockout/knockdown controls: When possible, use genetic deletion/silencing systems

Application-specific validation:
Different applications may require distinct validation approaches:

• For ELISA: Establish standard curves with purified Ara h 1 and determine limits of detection

• For immunohistochemistry: Include peptide blocking controls

• For flow cytometry: Use fluorescence-minus-one (FMO) controls

What considerations should be made when designing experiments using Ara h 1, clone P17 antibodies for different detection methods?

Successful implementation of Ara h 1, clone P17 antibodies requires careful experimental design tailored to specific detection methods:

Western blotting:

• Sample preparation: Consider native vs. reducing conditions (some epitopes may be conformation-dependent)

• Transfer efficiency: Optimize transfer parameters for high-molecular-weight oligomers

• Blocking agent: Avoid milk-based blockers which may contain cross-reactive proteins; use BSA instead

• Antibody concentration: Typically 1:500-1:2000 dilution for primary antibodies

• Controls: Include recombinant Ara h 1 (26-614aa) as positive control

ELISA:

• Coating buffer: Carbonate buffer (pH 9.6) typically yields optimal protein adsorption

• Blocking optimization: Test multiple blockers (BSA, casein, commercial blockers)

• Antibody pairs: For sandwich ELISA, select capture and detection antibodies recognizing non-overlapping epitopes

• Signal amplification: Consider avidin-biotin systems for enhanced sensitivity

• Standard curve: Use purified Ara h 1, clone P17 at 0.1-1000 ng/mL range

Immunofluorescence:

• Fixation method: Test multiple fixatives (PFA, methanol) as they may affect epitope accessibility

• Permeabilization: Optimize detergent concentration for intracellular access without destroying epitopes

• Antibody concentration: Typically higher than Western blot (1:100-1:500)

• Counterstains: Select nuclear and cytoskeletal markers compatible with experiment

• Controls: Include peptide competition controls to verify specificity

Flow cytometry:

• Cell preparation: Optimize fixation/permeabilization for intracellular staining

• Antibody titration: Determine optimal concentration to maximize signal-to-noise ratio

• Compensation: Account for spectral overlap when using multiple fluorophores

• Gating strategy: Develop consistent gating approach for identifying positive populations

• Controls: Include fluorescence-minus-one (FMO) and isotype controls

How can Ara h 1, clone P17 antibodies contribute to developing improved diagnostic tools for peanut allergy?

Ara h 1, clone P17 antibodies offer significant potential for advancing peanut allergy diagnostics:

Component-resolved diagnostics:

• Develop multiplex assays using antibodies against multiple peanut allergens (Ara h 1-17)

• Quantify allergen-specific IgE levels with higher precision than whole-extract tests

• Create standardized reference materials for consistent diagnostic cutoffs

Epitope-specific diagnostics:
Researchers have established epitope-based indirect ELISA using synthetic peptides spanning Ara h 1 linear epitopes . These assays can:

• Detect antibodies against specific immunodominant regions

• Potentially distinguish clinically relevant from cross-reactive sensitization

• Predict severity of allergic reactions based on epitope recognition patterns

Point-of-care testing:
Ara h 1 antibodies enable development of rapid lateral flow devices that:

• Use sandwich formats with matched antibody pairs

• Provide qualitative or semi-quantitative results

• Enable testing outside clinical laboratories

Clinical validation:
In one study, an epitope-based ELISA demonstrated 100% concordance with a commercial ELISA kit when testing 24 clinical serum samples from pigs , highlighting the potential for similar approaches in human diagnostics.

What role do Ara h 1, clone P17 antibodies play in understanding the immunopathogenesis of peanut allergy?

Ara h 1, clone P17 antibodies serve as crucial tools for investigating the complex immunological mechanisms underlying peanut allergy:

Cellular processing studies:

• Track intracellular routing of Ara h 1 in antigen-presenting cells

• Investigate how processing affects epitope presentation to T cells

• Examine differences in uptake between allergic and non-allergic individuals

T cell epitope discovery:
Tetramer Guided Epitope Mapping using Ara h 1 antibodies has revealed:

• Multiple T cell epitopes restricted by eight HLA class II alleles

• Higher frequency of Ara h 1-reactive T cells in allergic subjects (9 cells per million vs. <1 cell per million in non-allergic individuals)

• Distinct phenotypic profiles of allergen-specific T cells (CCR4+, CRTH2-, low β7 integrin expression)

Molecular mechanisms:
Antibody-based techniques have elucidated:

• Post-translational modifications affecting allergenicity (glycosylation, proteolytic processing)

• Role of N-terminal domain in oligomerization and enhanced allergenicity

• Contribution of both C-terminal and N-terminal domains to epitope formation

Cross-reactivity patterns:
Antibodies help map cross-reactive epitopes between:

• Different peanut allergens (Ara h 1-17)

• Homologous proteins in other legumes

• Structurally similar proteins in unrelated foods

These fundamental insights are essential for designing rational approaches to immunotherapy and identifying individuals at highest risk for severe reactions.

How can researchers use Ara h 1, clone P17 antibodies to evaluate novel immunotherapeutic approaches for peanut allergy?

Ara h 1, clone P17 antibodies provide essential tools for developing and evaluating new peanut allergy treatments:

Immunotherapy monitoring:

• Measure changes in allergen-specific antibody levels (IgE, IgG4) during treatment

• Track shifts in epitope recognition patterns as tolerance develops

• Assess blocking antibody activity against IgE binding

Modified allergen development:

• Verify structural integrity of hypoallergenic Ara h 1 variants

• Confirm epitope modification in engineered constructs

• Evaluate preservation of T cell epitopes while reducing IgE binding

Therapeutic mechanisms:

• Investigate allergen uptake and processing by tolerogenic dendritic cells

• Monitor Ara h 1-specific regulatory T cell induction during treatment

• Assess changes in basophil activation thresholds to Ara h 1 stimulation

Methodological approach:

• Establish baseline measurements of Ara h 1-specific immune parameters

• Apply immunotherapeutic intervention (oral, epicutaneous, modified allergens)

• Monitor changes in antibody levels, cellular responses, and epitope recognition

• Correlate immunological changes with clinical outcomes

The characterization of Ara h 1 T cell epitopes through tetramer-based techniques has provided critical knowledge that informs epitope-focused immunotherapy approaches, potentially allowing for more precise and effective interventions.