Major allergen Api g 1, isoallergen 1 Antibody

What is Api g 1 and how is it characterized molecularly?

Api g 1 is a major allergen found in celery (Apium graveolens) and belongs to the pathogenesis-related protein family PR-10, related to the Bet v 1 birch pollen allergen. It has a molecular mass of approximately 16 kDa and plays a critical role in the birch-celery syndrome . The open reading frame of Api g 1 cDNA encodes a protein of 153 amino acids with 40% identity (60% similarity) to Bet v 1 .

Api g 1 is recognized as a major allergen due to its high sensitization rate (75%) among celery-allergic individuals . The protein is located within the celeriac tuber (A. graveolens L. var. rapaceum), which is commonly found in processed food products like spice blends and soups .

Molecular characterization involves:

• SDS-PAGE analysis showing migration at approximately 16 kDa

• Isoelectric focusing to differentiate isoforms

• Mass spectrometry to confirm exact molecular weight and sequence

• Circular dichroism spectroscopy to analyze secondary structure

• Immunoblotting with specific antibodies or patient sera for allergenicity assessment

How do the isoforms of Api g 1 differ in structure and allergenicity?

Api g 1 exists in at least two distinct isoforms, Api g 1.0101 and Api g 1.0201, which show considerable differences in structure and allergenicity:

Studies have demonstrated that recombinant Api g 1.0101 exhibits stronger IgE-binding capacity compared to Api g 1.0201 in immunoblot tests with sera from celery-allergic patients . This difference in allergenicity is likely attributed to the specific amino acid variations between the isoforms, particularly the absence of leucine and the substitution of negatively charged glutamine with positively charged lysine in Api g 1.0101 .

These structural differences influence the conformation of IgE epitopes, affecting antibody recognition and the subsequent allergic response intensity, which has important implications for diagnostic test development and specificity .

What methods are effective for detecting Api g 1 in research settings?

Several complementary methods have proven effective for detecting Api g 1 in research settings:

Immunoassay-based detection:

• ELISA using specific antibodies can detect Api g 1 with high sensitivity (detection limits typically in the ng/mL range)

• Immunoblotting allows visualization of Api g 1 in complex protein mixtures and can distinguish between isoforms

• Protein microarrays enable multiplex allergen detection in a single sample

Mass spectrometry approaches:

• LC-MS/MS with multiple reaction monitoring for highly specific detection and quantification

• MALDI-TOF MS for rapid identification of Api g 1 in protein fractions

Molecular biological methods:

• qRT-PCR to detect Api g 1 gene expression in celery tissues

• Digital PCR for absolute quantification of Api g 1 genetic material

Extraction optimization:

• TCA/acetone extraction protocols show good recovery of Api g 1 from celery tissues

• Tris-HCl buffer (50mM, pH 7.4) with 150 mM NaCl and protease inhibitors, followed by protamine sulfate depletion (0.1%) for RuBisCO removal, improves purification of Api g 1

Research shows that detection sensitivity varies depending on the protein extraction method, with combined approaches using TCA/acetone precipitation after initial buffer extraction yielding the best results for subsequent immunodetection . For complex food matrices, additional cleanup steps may be necessary to remove interfering substances prior to Api g 1 detection.

How are high-quality antibodies against Api g 1 developed and characterized?

Development of high-quality antibodies against Api g 1 involves multiple strategic steps and rigorous characterization:

Immunization strategies:

• Recombinant protein immunization: Using highly purified recombinant Api g 1.0101 or Api g 1.0201 as immunogens

• Peptide immunization: Using unique peptide sequences from Api g 1 conjugated to carrier proteins

• DNA immunization: Expressing Api g 1 in vivo following plasmid injection

Antibody generation platforms:

• Polyclonal antibodies: Generated in rabbits or other suitable hosts using recombinant Api g 1 (amino acids 1-159)

• Monoclonal antibodies: Developed using hybridoma technology with B-cells from immunized mice

• Recombinant antibodies: Isolated from antibody libraries via phage display technology

Critical characterization steps:

• Affinity determination using surface plasmon resonance (SPR)

• Specificity evaluation through cross-reactivity testing with related allergens, particularly Bet v 1

• Epitope mapping to identify binding regions and potential cross-reactive epitopes

• Functional testing through immunoprecipitation assays with native allergens

• Performance evaluation in various immunoassay formats (ELISA, immunoblotting)

Research has shown that high-affinity recombinant antibodies against Api g 1 can be successfully generated using phage display technology from immunized mouse IgG libraries . These antibodies exhibit high affinity toward both recombinant and native Api g 1 from natural sources. Interestingly, while some Api g 1-specific antibodies cross-react with Bet v 1, others show no cross-reactivity despite the structural similarities between these allergens .

What methodological approaches enable effective epitope mapping of Api g 1?

Effective epitope mapping of Api g 1 requires sophisticated methodological approaches that can identify both linear and conformational epitopes:

Solution-phase techniques:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) identifies regions protected from solvent exchange upon antibody binding

• Nuclear magnetic resonance (NMR) spectroscopy detects chemical shift perturbations when antibodies interact with specific epitope regions

• Surface plasmon resonance (SPR) combined with mutant protein variants quantifies how specific amino acid substitutions affect binding kinetics

Solid-phase techniques:

• Synthetic peptide arrays containing overlapping sequences from Api g 1 identify linear epitopes

• Phage display libraries expressing Api g 1 fragments map both linear and conformational epitopes

• Alanine scanning mutagenesis identifies critical residues within epitopes

Computational approaches:

• Molecular modeling of Api g 1 structure based on homology to Bet v 1 (40% sequence identity)

• Epitope prediction algorithms that analyze surface accessibility and physico-chemical properties

• In silico docking simulations of antibody-Api g 1 interactions

Cross-reactivity studies:

• Cross-inhibition experiments between Api g 1 and Bet v 1 using specific antibodies and patient sera reveal shared epitopes

• Patient-specific IgE binding patterns indicate that the epitopes of cross-reactive allergens such as Api g 1 and Pru av 1 (cherry) are highly patient-specific

Research has demonstrated that the antibody response to Api g 1 is complex, with distinct epitope recognition patterns between patients. Methodologically, a combination of these approaches provides the most comprehensive epitope mapping, with SPR and immunoassay-based experiments offering quantitative data on binding characteristics and cross-reactivity .

How can Api g 1-specific antibodies be optimized for immunoassay development?

Optimizing Api g 1-specific antibodies for immunoassay development involves several strategic considerations and technical refinements:

Antibody pair selection:

• Screen multiple antibody clones to identify complementary pairs recognizing different epitopes

• Evaluate sandwich pairs that can simultaneously bind Api g 1 without steric hindrance

• Select antibodies with distinct isoform specificity (Api g 1.0101 vs. Api g 1.0201) or pan-reactivity based on assay requirements

Antibody engineering approaches:

• Affinity maturation through directed evolution or site-directed mutagenesis to improve binding characteristics

• Format conversion (IgG, Fab, scFv) to optimize assay performance and reduce non-specific binding

• Recombinant expression and purification to ensure batch-to-batch consistency

Optimization parameters:

• Buffer composition: Evaluate ionic strength, pH, detergents, and blocking agents to maximize signal-to-noise ratio

• Antibody immobilization strategies: Compare direct adsorption, oriented coupling, and biotin-streptavidin systems

• Detection conjugates: Test various enzyme labels (HRP, AP), fluorophores, or other detection systems

Validation criteria:

• Analytical sensitivity: Determine limits of detection and quantification in relevant matrices

• Specificity: Assess cross-reactivity with other allergens, particularly Bet v 1 and other PR-10 proteins

• Robustness: Evaluate performance with processed samples mimicking food processing conditions

Research demonstrates that high-affinity Api g 1-specific recombinant antibodies isolated by phage display technology show excellent performance in immunological assays, recognizing both recombinant allergens and native allergens from natural sources . Interestingly, some Api g 1-specific antibodies bind also to Bet v 1, while others show no cross-reactivity to Bet v 1 despite structural similarities, highlighting the importance of careful antibody selection for assay development .

How does Api g 1 cross-reactivity with Bet v 1 manifest at the molecular level?

Cross-reactivity between Api g 1 and Bet v 1 manifests through specific molecular mechanisms that explain the clinical phenomenon of the birch-celery syndrome:

Structural basis:

• Api g 1 shares approximately 40% sequence identity (60% similarity) with Bet v 1, the major birch pollen allergen

• Both proteins belong to the PR-10 family with conserved three-dimensional structures

• They share a similar fold consisting of a seven-stranded anti-parallel β-sheet and three α-helices forming a hydrophobic cavity

• X-ray crystallography data (PDB ID: 2BK0 for Api g 1.0101) reveals structural homology with Bet v 1

Immunological evidence:

• Cross-inhibition experiments demonstrate that pre-incubation with one allergen reduces IgE binding to the other, confirming shared epitopes

• Recombinant antibody studies have isolated Api g 1-specific antibodies that also recognize Bet v 1, providing direct evidence of common B-cell epitopes

• The reactivity of recombinant Api g 1 with IgE antibodies from celery-intolerant patients is comparable to that of natural celery allergen

Clinical correlation:

• Individuals sensitized to birch pollen frequently display type-I allergic symptoms after ingestion of celery, manifesting as the birch-celery syndrome

• This supports the hypothesis that allergies to certain vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen

Interestingly, while Bet v 1 is the primary sensitizing allergen in birch pollen, Api g 1 does not have the capability to sensitize and is lacking major T-cell epitopes, suggesting a unidirectional relationship where initial sensitization to Bet v 1 leads to subsequent cross-reactive responses to Api g 1 .

What structural features distinguish Api g 1 from other PR-10 food allergens?

Api g 1 possesses several distinctive structural features that differentiate it from other PR-10 food allergens:

Thermal stability profile:

• Unlike other Bet v 1 homologues such as Mal d 1 (apple) and Dau c 1 (carrot), Api g 1 can undergo structural changes at higher temperatures but uniquely return to its native structure after recooling

• This thermal reversibility potentially explains why some celery-allergic patients react to both raw and cooked celery, while heat-processed apple may be tolerated

Isoform characteristics:

• Api g 1 has two well-characterized isoforms (Api g 1.0101 and Api g 1.0201) with only 52% sequence identity between them, representing greater isoform diversity than some other PR-10 food allergens

• The isoforms show significant differences in IgE-binding capacity, with Api g 1.0101 exhibiting stronger allergenicity

Gastric stability:

• Api g 1 can acquire features of a complete allergen in hypoacidic gastric conditions, enabling sensitization via the gastrointestinal route

• This property is not universal among PR-10 proteins and may contribute to Api g 1's allergenic potency

Epitope organization:

• Api g 1 presents highly patient-specific IgE-binding epitopes, similar to Pru av 1 in cherries

• The surface distribution of these epitopes creates unique recognition patterns that differ from other PR-10 food allergens

Structural data availability:

• The three-dimensional structure of Api g 1.0101 has been determined (PDB ID: 2BK0) , providing precise structural information

• Comparative structural analysis reveals subtle differences in surface-exposed loops and the hydrophobic cavity that may influence allergenicity

These distinguishing features have significant implications for understanding the differential clinical reactivity patterns observed among patients allergic to various PR-10-containing foods and may explain why some individuals with birch pollen allergy react more strongly to celery than to other cross-reactive foods .

How do experimental conditions affect Api g 1 stability and antibody recognition?

Experimental conditions significantly influence both Api g 1 stability and antibody recognition, with important implications for research protocols and assay development:

pH effects:

• Api g 1 maintains stability across a moderate pH range (pH 5-8), but extremes can denature the protein

• Under hypoacidic gastric conditions, Api g 1 can retain its allergenicity and even acquire features of a complete allergen capable of sensitization

• Buffer selection for immunoassays should consider optimal pH for both allergen stability and antibody binding

Temperature considerations:

• Unlike some PR-10 proteins, Api g 1 has unique thermal properties, changing structure at higher temperatures but returning to its native conformation upon cooling

• Heating samples during extraction or processing may temporarily alter epitopes but not permanently destroy them

• Temperature cycling during analytical procedures should be carefully controlled to ensure consistent antibody recognition

Extraction protocols:

• Different extraction methods yield varying Api g 1 recovery and integrity

• TCA/acetone extraction and Tris-HCl buffer (pH 7.4) with protease inhibitors show effective extraction of Api g 1

• Protamine sulfate depletion (0.1%) can be used to remove abundant proteins like RuBisCO that may interfere with detection

Storage stability:

• Purified Api g 1 antibodies should be stored at -20°C or -80°C to maintain activity

• Repeated freeze-thaw cycles should be avoided by making aliquots

• Addition of stabilizers (e.g., glycerol) can enhance long-term antibody stability

Detection system variables:

• Antibody immobilization methods significantly affect orientation and binding capacity

• Blocking agents can influence background and specific signal intensity

• Detection conjugates (enzymes, fluorophores) may have different stability profiles

Experimental data shows that sample preparation is particularly critical, with immunoblotting results demonstrating that different protein extraction protocols yield varying band intensities for Api g 1 detection in celery tissues . For optimal results, freshly extracted proteins using appropriate buffers with protease inhibitors provide the most reliable detection, while maintaining consistent temperature and pH conditions throughout the analytical workflow ensures reproducible antibody recognition .

How can Api g 1 antibodies contribute to understanding pollen-food allergy syndrome mechanisms?

Api g 1 antibodies serve as powerful tools for investigating the complex mechanisms underlying pollen-food allergy syndrome through multiple research approaches:

Epitope mapping and cross-reactivity studies:

• Monoclonal or recombinant antibodies recognizing specific Api g 1 epitopes can identify shared structural features with Bet v 1

• Competitive binding assays using these antibodies quantify the degree of cross-reactivity between pollen and food allergens

• Epitope-specific antibodies help determine which protein regions are most important for cross-reactivity

Molecular visualization approaches:

• Immunohistochemistry with Api g 1 antibodies localizes the allergen within plant tissues, providing insights into allergen exposure

• Immunogold electron microscopy offers nanoscale resolution of allergen distribution

• Fluorescently-labeled antibodies enable tracking of allergen uptake and processing by immune cells

Allergen quantification in processed foods:

• Standardized immunoassays using Api g 1 antibodies measure allergen persistence during food processing

• Monitoring Api g 1 levels in various food preparations evaluates the relationship between allergen dose and clinical reactivity

• Detection of modified Api g 1 forms helps identify processing conditions that reduce allergenicity

Functional mechanistic studies:

• Api g 1 antibodies can block specific epitopes in cellular experiments to determine their functional importance

• Inhibition assays with basophils or mast cells from allergic patients demonstrate the biological relevance of cross-reactive epitopes

• Antibody-allergen immune complexes provide insights into how allergen presentation affects immune responses

Research has shown that high-affinity allergen-specific recombinant antibodies against Api g 1 with interesting binding properties can provide new preliminary insights to elucidate the mechanism behind the pollen-food syndrome . These antibodies demonstrate that despite similarities in Api g 1 and Bet v 1 tertiary structures, not all Api g 1-specific antibodies show cross-reactivity to Bet v 1, indicating epitope diversity that may explain patient-specific clinical manifestations .

What methodological approaches can differentiate between Api g 1 isoforms in research settings?

Differentiating between Api g 1 isoforms (Api g 1.0101 and Api g 1.0201) requires sophisticated methodological approaches that can detect subtle molecular differences:

Immunological methods:

• Isoform-specific monoclonal antibodies that recognize unique epitopes on each isoform

• Sandwich ELISA using strategically selected antibody pairs with differential recognition

• Lateral flow immunoassays with isoform-specific detection zones

Protein separation techniques:

• 2D-electrophoresis combining isoelectric focusing (IEF) with SDS-PAGE to separate isoforms based on both pI and molecular weight

• High-performance liquid chromatography (HPLC) with specifically optimized gradients

• Capillary electrophoresis for high-resolution separation of isoforms

Mass spectrometry approaches:

• Peptide mass fingerprinting following digestion to identify isoform-specific peptides

• Multiple reaction monitoring (MRM) targeting unique peptide sequences from each isoform

• Top-down proteomics analyzing intact proteins to distinguish between isoforms

Molecular techniques:

• PCR with isoform-specific primers to detect differential expression at the mRNA level

• Digital droplet PCR for absolute quantification of each isoform's transcript

• Next-generation sequencing to analyze the complete repertoire of Api g 1 isoforms

Structural analysis:

• Circular dichroism (CD) spectroscopy to detect subtle differences in secondary structure

• Differential scanning calorimetry to measure thermostability differences between isoforms

• Hydrogen-deuterium exchange mass spectrometry to map conformational differences

Research has demonstrated that the two isoallergens have 52% sequence identity with each other , and Api g 1.0101 generally exhibits stronger IgE-binding capacity compared to Api g 1.0201 . These differences are potentially attributable to key amino acid variations: Api g 1.0101 is missing a leucine residue, and has a positively charged lysine instead of a negatively charged glutamine when compared to Api g 1.0201 .

How can experimental designs address contradictory findings in Api g 1 cross-reactivity studies?

Addressing contradictory findings in Api g 1 cross-reactivity studies requires carefully designed experimental approaches that account for multiple variables:

Patient population stratification:

• Geographic stratification: Compare results from distinct geographic regions (e.g., Central European vs. Mediterranean populations)

• Sensitization profile analysis: Group patients based on primary sensitization patterns (birch pollen-primary vs. food-primary)

• Clinical phenotyping: Differentiate between patients with different symptom manifestations (OAS vs. systemic reactions)

Standardized allergen preparations:

• Use well-characterized recombinant allergens with verified folding and activity

• Include both isoforms (Api g 1.0101 and Api g 1.0201) in comparative studies

• Employ natural allergen extracts alongside recombinants to capture full complexity

Multi-method validation approach:

• Implement multiple complementary assays (ELISA, immunoblotting, basophil activation)

• Perform cross-inhibition experiments in both directions to establish relationship hierarchy

• Include appropriate positive and negative controls for each method

Statistical and analytical considerations:

• Calculate inhibition percentages rather than absolute values to normalize between patients

• Apply multivariate analysis to identify patterns across heterogeneous datasets

• Use hierarchical clustering to identify patient subgroups with similar reactivity patterns

Critical comparison framework:

• Directly compare seemingly contradictory studies using identical patient sera when possible

• Standardize reporting of methods and materials to facilitate cross-study comparison

• Employ meta-analysis approaches to synthesize findings across multiple studies

Research has revealed significant geographic variation in Api g 1 sensitization patterns. For example, a study comparing Swiss and French patients found that recombinant Api g 1 allowed accurate diagnosis of celery allergy in all Swiss patients, while only 2 of 12 French patients reacted to rApi g 1 . This suggests region-specific sensitization patterns that must be considered when designing and interpreting cross-reactivity studies. Additionally, sensitization patterns documented in a Mediterranean cohort showed that among 786 individuals tested, complex co-sensitization patterns existed between Api g 1, Art v 3 (mugwort pollen LTP), and Pru p 3 (peach LTP), with only some individuals displaying isolated Api g 1 reactivity .

How do Api g 1 antibodies contribute to developing improved diagnostic methods for celery allergy?

Api g 1 antibodies provide crucial tools for developing enhanced diagnostic methods for celery allergy through several innovative applications:

Standardized allergen quantification:

• Calibrated immunoassays using specific antibodies ensure consistent allergen content in diagnostic extracts

• Monoclonal antibody pairs in sandwich ELISA formats enable precise quantification of Api g 1 in standardized preparations

• Quality control systems employing antibody-based detection verify extract potency and stability

Novel diagnostic platforms:

• Multiplex microarray immunoassays incorporate Api g 1-specific antibodies alongside other food and pollen allergens for comprehensive patient profiling

• Lateral flow devices using high-affinity antibodies enable point-of-care testing

• Biosensor platforms with immobilized antibodies provide rapid, quantitative allergen detection

Component-resolved diagnostics:

• Antibodies distinguishing between Api g 1.0101 and Api g 1.0201 enable isoform-specific diagnosis

• Detection of recombinant and native forms provides complementary diagnostic information

• Parallel testing with Bet v 1 helps differentiate between primary celery sensitization and cross-reactivity

Clinical application insights:

These findings highlight the importance of region-specific diagnostic approaches. Research utilizing Api g 1-specific antibodies has revealed that while Api g 1 is a major allergen for Central European patients (sensitization rate 75%) , different geographical populations may react to distinct celery allergens. This knowledge allows for the development of regionally tailored diagnostic panels incorporating the most relevant allergen components for specific patient populations .

What are the methodological considerations for studying Api g 1 in processed food matrices?

Studying Api g 1 in processed food matrices presents unique methodological challenges that require specialized approaches:

Extraction optimization:

• Matrix-specific protocols are essential as food processing affects extractability

• Sequential extraction with multiple buffers may be necessary to recover modified Api g 1 forms

• Addition of detergents (SDS, Tween) or reducing agents may improve recovery from complex matrices

• Inclusion of protease inhibitors prevents degradation during extended extraction procedures

Detection method considerations:

• Antibody selection should account for potential processing-induced epitope modifications

• Pair antibodies recognizing different epitopes to increase chances of detection after processing

• Consider multiple antibody clones with varying specificity to capture different Api g 1 forms

Processing effect characterization:

• Thermal stability studies examining Api g 1's unique ability to refold after cooling, unlike other PR-10 allergens

• pH stability assessment, particularly relevant as Api g 1 can acquire complete allergen features under hypoacidic conditions

• Matrix interaction studies to determine how other food components affect Api g 1 stability and extractability

Analytical validation requirements:

• Include matrix-matched standard curves to account for matrix effects

• Perform spike-recovery experiments at multiple concentrations

• Utilize incurred samples (naturally containing Api g 1) alongside spiked samples

• Compare multiple detection methods (immunological, mass spectrometry) for confirmation

Reference materials development:

• Create matrix-specific reference materials containing known Api g 1 concentrations

• Process reference materials under standardized conditions to mimic commercial processing

• Characterize reference materials using orthogonal methods to establish true Api g 1 content

Research has demonstrated that Api g 1 responds differently to thermal processing compared to other Bet v 1 homologues - it changes structure at higher temperatures compared to some homologues like Mal d 1 (apple) and Dau c 1 (carrot), but uniquely can return to its native structure after recooling . This property has significant implications for detection methodologies, as brief heating during sample preparation may temporarily alter epitopes without permanently destroying them, potentially leading to false negatives if sampling timing is not optimized.