Allergen Ara h 1, clone P17 Antibody, Biotin conjugated

What is Allergen Ara h 1, clone P17 and why is it significant in 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. The allergenicity of Ara h 1 has been linked to the specific arrangement of monomers in the homotrimeric structure of vicilin/7S globulin proteins . Peanut allergies mediated by immunoglobulin E (IgE) sensitization to these proteins represent a significant and potentially life-threatening health concern, making Ara h 1 detection and characterization critical for allergy research.

Methodological approach: Researchers typically use purified natural or recombinant forms of Ara h 1 in immunological assays to study its allergenic properties, epitope mapping, and cross-reactivity with other allergens.

What are the key characteristics of the Allergen Ara h 1, clone P17 Antibody, Biotin conjugated?

The Allergen Ara h 1, clone P17 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbits against recombinant Peanut Allergen Ara h 1, clone P17 protein (specifically amino acids 26-216) . Its key characteristics include:

What are the optimal storage conditions for this antibody?

For maximum stability and performance of the Allergen Ara h 1, clone P17 Antibody, Biotin conjugated:

• Upon receipt, the antibody should be shipped at 4°C

• For long-term storage, aliquot and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which can denature the antibody and reduce its binding efficacy

• Working solutions can be stored at 4°C for short periods but should be prepared fresh for optimal results

How should researchers optimize ELISA protocols using this biotin-conjugated antibody?

When designing ELISA protocols using Allergen Ara h 1, clone P17 Antibody, Biotin conjugated, researchers should consider the following methodological approaches:

• Capture antibody selection: Pair with an appropriate capture antibody such as anti-Ara h 1 monoclonal antibody 2C12 (as used in commercial kits)

• Detection system optimization:

  • Use streptavidin-HRP conjugate for signal development

  • Optimize the concentration of biotin-conjugated antibody (typically starting with 1:1000 dilution)

  • Use 3,3',5,5'-Tetramethylbenzidine (TMB) as substrate for color development

• Blocking and wash steps:

  • Block plates with 1-3% BSA in PBS containing 0.1% Tween (PBST)

  • Include multiple washing steps with PBST between incubations

  • Consider automated washing for consistency

• Sample preparation:

  • For food samples: Optimize extraction buffers to maximize allergen recovery

  • For serum samples: Consider pretreatment to minimize matrix effects

• Standard curve development:

  • Use purified natural or recombinant Ara h 1 for calibration

  • Commercial kits typically use a range of 2,000 to 4.0 ng/mL

How can researchers evaluate potential cross-reactivity between Ara h 1 and other peanut allergens?

Cross-reactivity assessment requires careful experimental design:

• Inhibition ELISA methodology:

  • Pre-incubate serum with serial dilutions of purified allergens (rAra h 1, 2, 3, etc.)

  • Add the pre-incubated serum to ELISA plates coated with target allergen

  • Measure reduction in IgE binding compared to uninhibited controls

  • Analyze inhibition curves starting at low concentrations (approximately 1 ng/mL)

• Reducing vs. non-reducing conditions:

  • Compare antibody binding under reducing conditions (using DTT) and non-reducing conditions

  • Significant decrease in binding under reducing conditions may indicate disulfide-linked contamination with other allergens rather than true cross-reactivity

• Mass spectrometry validation:

  • Use LC-MS/MS to verify purity of allergen preparations

  • Identify potential contaminating proteins that could cause false positive cross-reactivity results

• Cross-contamination control:

  • Use both natural purified and recombinant allergens to distinguish between true cross-reactivity and contamination

  • Employ synthetic peptides representing postulated cross-reactive epitopes

What analytical techniques can confirm the purity and specificity of allergen preparations?

Multiple complementary methods should be used:

• SDS-PAGE and Western blotting:

  • Run samples under reducing and non-reducing conditions

  • Probe with Ara h 1-specific and Ara h 2-specific monoclonal antibodies to detect potential contamination

  • Look for higher molecular mass bands that may indicate protein complexes

• Sandwich ELISA:

  • Develop specific sandwich ELISAs using antibodies against different allergens

  • Quantify potential contaminants in purified preparations

  • Detection limits as low as 31.5 ng/mL can be achieved with optimized systems

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS):

  • Identify and quantify allergen components and contaminants

  • Can detect <1% contamination of Ara h 2 and Ara h 6 in purified Ara h 1 and Ara h 3 preparations

• Circular dichroism (CD):

  • Verify conformational integrity of allergen preparations

  • Useful for confirming structural changes during probe-target interactions

How can this antibody be utilized in T-cell epitope identification studies?

While the biotin-conjugated antibody itself is primarily used for protein detection, it can be incorporated into comprehensive T-cell epitope research workflows:

• Tetramer Guided Epitope Mapping (TGEM) methodology:

  • Use biotinylated HLA-DR proteins loaded with overlapping peptides spanning the Ara h 1 sequence

  • Generate peptide tetramers for T-cell detection

  • Culture cells for 14 days and then stain with pooled peptide tetramers

  • Identify positive wells and restain using tetramers loaded with individual peptides

• Epitope identification workflow:

  • Synthesize overlapping peptides (e.g., 20 amino acids with 12 amino acid overlap) spanning the Ara h 1 sequence

  • Organize peptides into pools for initial screening

  • Use biotin-conjugated antibody to confirm epitope identity in downstream applications

  • Identify restriction elements using specific HLA class II alleles

• T-cell frequency determination:

  • Use anti-PE magnetic beads to enrich for PE-labeled tetramer-positive cells

  • Characterize phenotype and frequency of Ara h 1-reactive T cells without in vitro expansion

  • Compare frequencies between allergic and non-allergic individuals (approximately 9 cells per million in allergic subjects versus less than 1 cell per million in non-allergic subjects)

What phenotypic markers are associated with Ara h 1-reactive T cells in allergic individuals?

Research has identified specific phenotypic characteristics of Ara h 1-reactive T cells:

• Surface marker expression:

  • Ara h 1-reactive T cells in allergic subjects express CCR4

  • These cells do not express CRTH2

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

• Cytokine profiles:

  • Ara h 1-reactive cells secrete multiple cytokines including:

    • IFN-γ

    • IL-4

    • IL-5

    • IL-10

    • IL-17

• Frequency analysis:

  • Higher frequency of Ara h 1-reactive T cells in allergic versus non-allergic individuals

  • Detection methodology involves ex vivo enrichment using PE-labeled tetramers

What molecular mechanisms explain cross-reactivity between Ara h 1 and other peanut allergens?

Despite low sequence identities between major peanut allergens, cross-reactivity has been observed due to several mechanisms:

• Disulfide interactions:

  • Highly purified natural Ara h 1 (nAra h 1) may contain small amounts (<1%) of Ara h 2 and Ara h 6 contamination

  • These contaminants can be covalently bound to Ara h 1 via disulfide interactions

  • Cross-reactivity is lost when purified nAra h 1 is pretreated under reducing conditions, suggesting that these contaminants are responsible for apparent cross-reactivity

• Detection of contamination:

  • SDS-PAGE/Western blot analysis using Ara h 2-specific monoclonal antibodies can detect Ara h 2 contamination in purified nAra h 1

  • Reducing conditions decrease the binding of Ara h 2-specific monoclonal antibodies to nAra h 1 by approximately 77%

• Experimental validation:

  • IgE cross-inhibition between 2S albumins and Ara h 1 is observed only with natural purified allergens, not with recombinant allergens or synthetic peptides

  • This suggests that true cross-reactivity at the epitope level is minimal

How can researchers distinguish between natural contamination and true cross-reactivity?

Methodological approaches to differentiate contamination from true cross-reactivity include:

• Comparison of natural and recombinant allergens:

  • Use both natural purified allergens and recombinant forms in inhibition assays

  • True epitope cross-reactivity would be observed with both forms

  • Contamination-based apparent cross-reactivity would only be observed with natural purified allergens

• Reducing condition experiments:

  • Pre-treat allergen preparations with reducing agents like DTT

  • Wash extensively to remove dissociated contaminants

  • Compare binding before and after reduction

  • Significant decrease in binding after reduction suggests contamination rather than true cross-reactivity

• Synthetic peptide studies:

  • Use synthetic peptides representing postulated cross-reactive epitopes

  • Test in inhibition assays alongside natural and recombinant allergens

  • Lack of inhibition with synthetic peptides supports contamination hypothesis

• Advanced analytical techniques:

  • Combine sandwich ELISA, Western blotting, and LC-MS/MS for comprehensive characterization

  • Quantify contamination levels in allergen preparations

  • Establish threshold levels for potential contribution to apparent cross-reactivity

What are the critical controls needed when designing experiments with this antibody?

Robust experimental design requires several types of controls:

• Antibody specificity controls:

  • Negative control: Incubate with buffer instead of primary antibody

  • Isotype control: Use non-specific rabbit IgG at the same concentration

  • Cross-reactivity control: Test against related but distinct allergens

• ELISA-specific controls:

  • Background control: Coat wells with blocking solution only

  • Standard curve: Include purified Ara h 1 at known concentrations (2,000-4.0 ng/mL range)

  • Limit of detection determination: Include low concentration samples near the detection threshold (approximately 31.5 ng/mL)

• Inhibition assay controls:

  • Self-inhibition control: Pre-incubate with the same allergen as the coating

  • Expected homologous inhibition should reach 75-95% at around 1 μg/mL

  • Non-related allergen control: Include an unrelated allergen as negative control

• Reduction controls:

  • Compare binding under reducing and non-reducing conditions

  • Include controls for antibody sensitivity to reduction itself

  • Ensure that washing steps effectively remove any released contaminants

How can researchers optimize sensitivity in detection assays using this antibody?

To maximize sensitivity in Ara h 1 detection:

• Signal amplification strategies:

  • Leverage the biotin-streptavidin system's high affinity

  • Use streptavidin-HRP with optimized substrate systems

  • Consider tyramide signal amplification for ultra-sensitive detection

• ELISA optimization:

  • Titrate antibody concentrations to determine optimal working dilution

  • Evaluate different blocking agents (BSA, casein, commercial blockers)

  • Optimize incubation times and temperatures

  • Use automated washing to ensure consistency

• Sample preparation refinement:

  • Optimize extraction buffers for food matrices

  • Consider sample concentration methods for low-abundance samples

  • Address potential matrix effects that may interfere with detection

• Detection system selection:

  • Choose appropriate substrate based on sensitivity requirements

  • TMB substrate offers good sensitivity for colorimetric detection

  • Consider chemiluminescent substrates for enhanced sensitivity

What approaches can resolve discrepancies between different detection methods?

When facing inconsistent results across detection platforms:

• Comparative analysis protocol:

  • Run samples in parallel using multiple detection methods

  • Include SDS-PAGE/Western blot, ELISA, and mass spectrometry

  • Analyze under both reducing and non-reducing conditions

• Potential sources of discrepancy:

  • Epitope accessibility differences between methods

  • Matrix effects specific to certain detection platforms

  • Different detection limits and dynamic ranges

  • Conformational versus linear epitope detection

• Resolution strategies:

  • Use recombinant allergens and synthetic peptides as reference standards

  • Implement spike-and-recovery experiments to assess matrix effects

  • Develop correction factors based on systematic comparative studies

  • Combine multiple methods for comprehensive characterization

• Standardization approaches:

  • Establish internal reference standards

  • Participate in inter-laboratory comparisons

  • Develop standardized protocols with defined acceptance criteria

  • Document method limitations and appropriate applications