Allergen Ara h 1, clone P17 Antibody, FITC conjugated

What is Allergen Ara h 1, clone P17 and what is its significance in peanut allergy research?

Allergen Ara h 1, clone P17 is a variant of the major peanut allergen Ara h 1 found in Arachis hypogaea (peanuts). Ara h 1, along with Ara h 2 and Ara h 3, contributes significantly to peanut allergies which are among the most severe food allergies . The allergenicity of Ara h 1 has been linked to the specific arrangement of monomers in the homotrimeric structure of vicilin/7S globulin proteins .

Ara h 1 occurs in different molecular forms, with the full 63 kDa glycosylated form being well-characterized. Research has also identified a 54 kDa truncated variant where the N-terminal domain has been removed . This protein is encoded by the gene with UniProt ID P43237 .

Understanding Ara h 1's molecular structure, epitope distribution, and immunogenic properties is crucial for developing diagnostic tools and therapeutic approaches for peanut allergies.

What are the optimal applications for Allergen Ara h 1, clone P17 Antibody, FITC conjugated?

The FITC-conjugated Allergen Ara h 1, clone P17 Antibody is primarily used in immunofluorescence-based detection methods. According to product specifications, this antibody has been tested and validated for ELISA applications . The fluorescein isothiocyanate (FITC) conjugation makes it particularly suitable for:

• Immunofluorescence microscopy

• Flow cytometry

• Multiplex immunoassays

• Fluorescence-based ELISA

While the antibody has been primarily tested in ELISA, researchers should validate its performance for other applications through appropriate controls. The antibody shows confirmed reactivity with Arachis hypogaea, making it suitable for peanut allergen detection in various experimental contexts .

What are the storage and handling recommendations for maintaining antibody activity?

For optimal preservation of antibody activity, the following conditions are recommended:

• Storage temperature: Store at -20°C or -80°C

• Form: The antibody is supplied in liquid form

• Buffer composition: 0.03% Proclin 300 as preservative, 50% Glycerol, 0.01M PBS, pH 7.4

• Avoid repeated freeze-thaw cycles by making aliquots upon receipt

• Briefly centrifuge tubes before opening to prevent loss of material adhering to caps or tube walls

These storage conditions are designed to maintain antibody stability and activity. The high glycerol content (50%) helps prevent freezing damage to the antibody protein structure, while the preservative (Proclin 300) inhibits microbial growth without affecting antibody function.

How do the molecular variants of Ara h 1 affect epitope recognition and experimental design?

Research has identified at least two important variants of Ara h 1:

• The full-length 63 kDa Ara h 1 - Contains both N-terminal and C-terminal domains

• A truncated 54 kDa variant - Lacks the N-terminal domain (starting with the amino acid sequence EGREGEQ-)

These variants display significant structural and functional differences:

The truncated 54 kDa variant has been found to occur "exclusively as a homotrimer, indicating that the N-terminal domain of the 63 kDa molecule may be involved in higher order oligomerization" . When designing experiments, researchers should consider:

• Which variant(s) they are targeting

• Whether the antibody's immunogen (aa 26-216) includes epitopes present in both variants

• The potential impact of oligomeric state on epitope accessibility

• The need for denaturation to expose hidden epitopes

For comprehensive allergen detection, methods targeting both variants may be necessary.

What is the role of glycosylation in Ara h 1 allergenicity and how can it be studied?

Glycosylation plays a crucial role in the allergenicity of Ara h 1. Research has shown that:

• Both the 63 kDa and 54 kDa Ara h 1 subunits contain N-linked glycans

• Two main types of N-glycans have been identified: high-mannose type and β-xylosylated type

• In at least one peanut allergy patient, "the cross-reactivity of IgE against Ara h 1 was completely lost by de-N-glycosylation, indicating the N-glycan of Ara h 1 was the sole epitope for the Ara h 1-specific IgE"

To study the role of glycosylation using FITC-conjugated Ara h 1 antibodies, researchers can:

• Compare native and deglycosylated Ara h 1 recognition using fluorescence-based assays

• Perform competitive binding studies with purified glycan moieties

• Use lectin co-localization studies alongside the FITC-labeled antibody

• Employ site-directed mutagenesis of glycosylation sites to determine critical glycan positions

These approaches can help distinguish between protein epitope recognition and glycan-dependent recognition, providing insights into the mechanism of allergenicity.

How can Allergen Ara h 1 antibodies be validated for specificity in experimental protocols?

Ensuring antibody specificity is critical for reliable research outcomes. For Allergen Ara h 1, clone P17 Antibody validation, researchers should consider:

Western Blot Validation:

• Expected molecular weight: 70 kDa (apparent MW)

• Sample preparation: Denaturing conditions with DTT (50 mM), heating at 70°C for 5 min

• Gel system: Tricine or similar gradient gels suitable for proteins in the 50-70 kDa range

• Blocking: 5% milk in PBST (PBS with 0.1% Tween-20)

• Antibody dilution: 1:1000 for primary antibody

ELISA Validation:

• Recommended dilution: 1:320,000 for indirect ELISA

• Controls should include:

  • Positive control: Purified recombinant Ara h 1

  • Negative control: Unrelated plant proteins

  • Secondary antibody-only control

  • Isotype-matched irrelevant antibody control

Cross-reactivity Assessment:
Test against other peanut allergens (Ara h 2-17) and homologous proteins from other legumes to confirm specificity.

What methodological considerations are important when using FITC-conjugated antibodies for multiparameter analysis?

When employing FITC-conjugated Ara h 1 antibodies in multiparameter analyses, several methodological considerations are critical:

Spectral Properties:

• FITC excitation maximum: ~495 nm

• FITC emission maximum: ~520 nm

• Consider spectral overlap with other fluorophores in multiplex experiments

Photobleaching Prevention:

• Minimize exposure to light during storage and experiment preparation

• Use anti-fade mounting media for microscopy applications

• Consider acquiring FITC channel data first in sequential acquisition protocols

Signal Optimization:

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Account for autofluorescence, particularly in plant tissue samples

• Perform compensation when combining with other fluorophores

Application-Specific Considerations:
For flow cytometry:

• Use appropriate cell fixation methods that preserve both antibody binding sites and fluorophore activity

• Include unstained and single-stained controls for accurate compensation

For microscopy:

• Select appropriate filter sets to maximize FITC signal collection while minimizing bleed-through

• Use sequential scanning for confocal applications to minimize crosstalk

What is the optimal protocol for using Allergen Ara h 1, clone P17 Antibody, FITC conjugated in Western blotting?

While the FITC-conjugated antibody is not the primary choice for Western blotting (HRP conjugates are typically preferred), it can be used with a fluorescence imaging system. Based on validated protocols for the non-conjugated version, the following approach can be modified for the FITC conjugate:

Sample Preparation:

• Extract proteins from defatted lightly roasted peanut flour with borate buffered saline (BBS) solution (100 mM H₃BO₄, 25 mM Na₂B₄O₇, 75 mM NaCl, pH 8.6) for 1 hour with constant stirring at 4°C

• Denature samples with LDS sample buffer containing 50 mM DTT (1:4 v/v ratio) at 70°C for 5 minutes

Gel Electrophoresis and Transfer:

• Separate proteins on 10-20% Tricine gels or similar gradient gels

• Transfer to nitrocellulose membrane using appropriate transfer system

Immunodetection (Modified for FITC):

• Block membrane with 5% milk in PBST (PBS with 0.1% Tween-20) for 1 hour at room temperature

• Incubate with FITC-conjugated primary antibody at 1:1000 dilution in PBST for 1 hour at room temperature

• Wash 3 times with PBST, 5 minutes each

• Image directly using a fluorescence imaging system with appropriate filters for FITC

Notes:

• Protect membrane from light during and after antibody incubation

• No secondary antibody is needed as the primary is directly conjugated

• Expected band: approximately 70 kDa

How can the Allergen Ara h 1, clone P17 Antibody be used to investigate epitope-specific immune responses?

The clone P17 antibody targets the region encompassing amino acids 26-216 of Ara h 1 . This region is particularly important for investigating epitope-specific immune responses because:

• It contains multiple B-cell epitopes relevant to peanut allergies

• It includes portions of both the N-terminal domain and core protein structure

For epitope mapping and characterization studies:

Competitive ELISA Approach:

• Coat plates with recombinant Ara h 1

• Pre-incubate patient sera with synthesized peptides representing different regions of Ara h 1 (26-216)

• Add the pre-incubated sera to the plates

• Detect bound IgE using appropriate secondary antibodies

• Compare inhibition patterns to identify immunodominant epitopes

Epitope-Resolved Assays:

• Generate a panel of peptides covering the 26-216 region

• Coat each peptide onto a separate well/bead

• Incubate with patient sera

• Detect bound IgE using appropriate secondary antibodies

• Use the FITC-conjugated Ara h 1 antibody to confirm full-length protein recognition

These approaches allow researchers to correlate epitope recognition patterns with clinical symptoms and develop more precise diagnostic tools for peanut allergies.

How does oligomeric structure affect Ara h 1 detection and antibody binding?

The oligomeric structure of Ara h 1 significantly impacts detection and antibody binding. Research has shown that:

• The 63 kDa Ara h 1 forms higher-order oligomeric structures (decamers or nonamers)

• The 54 kDa variant (lacking the N-terminal domain) exists exclusively as a homotrimer

• The N-terminal domain appears to be involved in higher-order oligomerization

These structural differences have important implications for antibody binding:

For optimal detection:

• Consider using mild detergents to partially disrupt higher-order structures

• Compare native and denaturing conditions to ensure complete epitope detection

• When quantifying Ara h 1, account for potentially different antibody affinities to various oligomeric forms

• For structural studies, use size exclusion chromatography in conjunction with antibody detection

Understanding these structural variations is particularly important for developing sensitive allergen detection methods for clinical and food safety applications.

What are the best practices for using FITC-conjugated antibodies in immunofluorescence microscopy for allergen localization?

For optimal allergen localization using FITC-conjugated Ara h 1 antibodies in immunofluorescence microscopy:

Sample Preparation:

• Fix tissue sections using 4% paraformaldehyde to preserve protein structure while maintaining antibody accessibility

• Perform antigen retrieval if necessary (heat-induced in citrate buffer, pH 6.0)

• Block with 5-10% normal serum from the same species as the secondary antibody (if used) or BSA

Immunostaining Protocol:

• Apply FITC-conjugated Ara h 1 antibody at optimized dilution (start with 1:100 and titrate)

• Incubate in a humidified chamber at 4°C overnight or room temperature for 1-2 hours

• Wash thoroughly with PBS (3 × 5 minutes)

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium specifically formulated for fluorescence preservation

Technical Considerations:

• Prepare negative controls by omitting primary antibody or using isotype controls

• Include positive controls using known Ara h 1-containing samples

• Minimize exposure to light throughout the protocol

• Consider co-localization studies with markers for specific cellular compartments

• For quantitative analysis, standardize exposure settings and acquisition parameters

Advanced Applications:

• Combine with other fluorescently-labeled antibodies (using different fluorophores) for multi-protein localization

• Consider super-resolution microscopy techniques for detailed subcellular localization of allergens

• For in vivo studies, consider two-photon microscopy which may reduce photobleaching of FITC

Following these best practices will ensure reliable and reproducible localization of Ara h 1 in tissue samples, providing valuable insights into allergen distribution and processing.

How do different conjugates of Ara h 1 antibodies compare in research applications?

Different conjugates of Allergen Ara h 1 antibodies offer distinct advantages for specific research applications:

For quantitative comparisons:

• HRP-conjugated antibodies typically offer 5-10x higher sensitivity than FITC in ELISA applications due to enzymatic signal amplification

• FITC-conjugated antibodies allow for direct visualization without additional reagents, simplifying protocols

• Unconjugated primary antibodies with labeled secondary antibodies can provide 2-4x signal amplification compared to directly conjugated primaries

When selecting a conjugate, researchers should consider:

• Required sensitivity

• Need for multiplexing

• Available detection equipment

• Sample autofluorescence (particularly relevant for plant tissues)

• Quantitative vs. qualitative analysis requirements

What emerging technologies are enhancing the utility of fluorescently-labeled allergen antibodies?

Several cutting-edge technologies are expanding the applications of fluorescently-labeled allergen antibodies like FITC-conjugated Ara h 1:

Advanced Imaging Technologies:

• Light-sheet microscopy for 3D visualization of allergen distribution in intact food matrices

• Super-resolution microscopy (STORM, PALM) to visualize allergen localization beyond the diffraction limit

• Correlative light and electron microscopy (CLEM) to combine ultrastructural information with specific allergen labeling

Microfluidic and Lab-on-a-Chip Systems:

• Integrated allergen detection platforms combining sample preparation and fluorescence detection

• Droplet-based microfluidic systems for high-throughput single-cell analysis of allergen-immune cell interactions

• Paper-based immunofluorescence assays for point-of-care allergen detection

Computational and AI-Enhanced Analysis:

• Machine learning algorithms for automated quantification of allergen distribution patterns

• Computational modeling of antibody-allergen interactions to predict cross-reactivity

• Image analysis software for co-localization studies with multiple allergens

Novel Conjugation Approaches:

• Quantum dot conjugation for increased photostability and brightness

• pH-sensitive fluorophores to track allergen processing through cellular compartments

• Click chemistry-based modular conjugation systems allowing flexible labeling strategies

These technologies provide researchers with unprecedented tools to study allergen structure, distribution, and interaction with the immune system, potentially leading to improved diagnostic methods and therapeutic approaches for peanut allergies.