Pru p 1.01 Antibody

What is Pru p 1.01 and why are antibodies against it significant for research?

Pru p 1.01 (Pru p 1.0101) is a major isoallergen found in peach and nectarine fruits belonging to the pathogenesis-related protein-10 (PR-10) family. It is homologous to the birch pollen allergen Bet v 1 and shows significant cross-reactivity with other PR-10 proteins . Antibodies against Pru p 1.01 are crucial for:

• Quantifying allergen levels in different peach varieties

• Studying cross-reactivity between related food allergens

• Investigating allergen distribution in various fruit tissues

• Understanding the structural and functional properties of this protein

Pru p 1.01 has a molecular weight of approximately 19.49 kDa and possesses ribonuclease (RNase) activity, which represents a common defense mechanism in plants responding to pathogen infection .

How are monoclonal and polyclonal antibodies against Pru p 1.01 typically produced?

Production of anti-Pru p 1.01 antibodies follows these methodological steps:

For monoclonal antibodies:

• Bal b/c mice are immunized subcutaneously and intraperitoneally with 10-40 μg of recombinant Pru p 1.0101 (rPru p 1.0101)

• Mice receive booster immunizations five times at 2-week intervals with 50 μg antigens in Incomplete Freund's adjuvant

• Antibody-producing hybridoma cells are selected through ELISA and Western blot screening

• Antibodies are purified using HiTrap Protein-A affinity chromatography

For polyclonal antibodies:

• Rabbits are immunized with recombinant Pru p 1.01

• Serum is collected after multiple immunizations

• Antibodies are purified from serum using standard methods

In one documented study, this approach yielded five monoclonal antibodies and two polyclonal antibodies with high specificity to their target antigens .

What considerations are important when designing a sandwich ELISA for Pru p 1 detection?

Developing an effective sandwich ELISA (sELISA) for Pru p 1 detection requires careful consideration of several methodological factors:

Antibody selection and validation:

• Choose monoclonal antibodies with high binding capacity and specificity as capturing antibodies

• Validate polyclonal antibodies as detection antibodies to recognize multiple epitopes

• Test combinations to determine optimal pairing (as shown in Table 1)

Pair matching example from research:

Note: Values represent optical density; higher values indicate better pair matching

Protocol optimization:

• Establish working range and detection limits (4-32 ng/ml is reported as effective)

• Determine appropriate sample dilutions based on tissue type (peel 1:100, pulp 1:5, whole fruit 1:20)

• Use recombinant Pru p 1 (mixture of isoforms) as a standard

• Validate assay precision, accuracy, reproducibility and specificity

How should isotype controls be integrated into Pru p 1.01 antibody experiments?

Isotype controls are essential negative controls in antibody-based experiments involving Pru p 1.01 detection:

Selection criteria:

• Choose isotype controls with the same constant heavy chain as the primary antibody

• Ensure the control lacks specific binding to Pru p 1.01 or related proteins

• Match critical properties including concentration, species, and isotype class

Implementation methodology:

• Run isotype controls under identical experimental conditions as the test antibody

• Use the same concentration, incubation parameters, blocking solutions, and detection methods

• Apply isotype controls to determine the level of non-specific background activity

• Use the background signal to normalize and interpret the true signal of the Pru p 1.01 interaction

Analysis considerations:

• Compare signal from primary antibody to isotype control run under the same conditions

• Minimal staining indicates low background interference

• Significant isotype control signal reveals background level that must be subtracted from test results

Note that while isotype controls reveal background staining, they don't independently confirm antibody specificity or indicate the source of background signals.

What methods can be used to purify and characterize natural Pru p 1?

Purification and characterization of natural Pru p 1 (nPru p 1) involves several specialized techniques:

Purification protocol:

• Affinity chromatography using specific monoclonal antibodies (such as A0-A7-G11)

• Separation of the purified protein by SDS-PAGE

• Confirmation of identity by Western blot showing a characteristic band at 17 kDa

Characterization methods:

• Mass spectrometry (LC-MS) for sequence verification

  • Reported sequence coverage: 75.0% for Pru p 1.0101 and 83.1% for Pru p 1.0201

• Immunoblotting with specific antibodies to confirm identity

• Functional assays such as RNase activity tests

Structural analysis:

• NMR solution structure determination

• Analysis of ligand binding (e.g., zeatin binding studies)

• Conformational stability assessments

• Evaluation of structural flexibility using NMR relaxation dispersion (RD) experiments

How can antibody specificity to different Pru p 1 isoallergens be evaluated?

Evaluating antibody specificity to different Pru p 1 isoallergens requires systematic testing:

Cross-reactivity assessment:

• Test antibodies against recombinant versions of each isoallergen (Pru p 1.0101, Pru p 1.0201, Pru p 1.0301)

• Use ELISA with pure recombinant proteins to quantify binding affinities

• Perform Western blot analysis to confirm recognition of specific protein bands

Example of specificity testing from published research:

Note: Values represent optical density; higher values indicate stronger binding

Competition assays:

• Perform competitive ELISA where unlabeled isoallergens compete with labeled ones

• Calculate inhibition percentages to determine relative affinities

• Compare epitope recognition patterns between different antibodies

How do researchers interpret variations in Pru p 1 content across different peach varieties?

Interpretation of Pru p 1 content variation requires considering multiple factors:

Methodological approach:

• Sandwich ELISA with validated antibodies measures total Pru p 1 levels

• Western blot confirms specific protein bands

• qPCR of Pru p 1 genes correlates protein levels with gene expression

Observed variations:

• Pru p 1 is mainly concentrated in the peel (0.20-73.44 μg/g fresh weight)

• Pulp contains significantly lower levels (0.05-9.62 μg/g)

• Wild peach varieties often show undetectable levels

• Whole fruit content varies widely (0.12-6.45 μg/g) across 78 peach and nectarine varieties

Interpretation frameworks:

• Genetic factors: Different cultivars express varying levels of Pru p 1 genes

• Developmental factors: Maximum expression occurs during the pit hardening phase

• Environmental influences: Fruit bagging during growth significantly reduces Pru p 1 gene expression

• Ripening stage: Late-ripening yellow flesh varieties typically contain higher allergen levels

Research shows that wild peach varieties and certain nectarines (such as "Zi Xue Tao," "Wu Yue Xian," and "May Fire") contain low levels of both Pru p 1 and Pru p 3 allergens, making them potential hypoallergenic options .

How does Pru p 1.01 antibody binding correlate with the protein's functional properties?

Understanding the relationship between antibody binding and Pru p 1.01 functional properties provides critical insights:

Functional activities of Pru p 1.01:

• Possesses ribonuclease (RNase) activity as a defense mechanism

• Interacts with ligands such as zeatin

• Shows structural flexibility in specific regions

Correlative analysis methods:

• Compare antibody binding regions with functional domains

• Assess whether antibody binding affects enzymatic activity

• Evaluate changes in protein conformation upon antibody binding

Research observations:

• Zeatin inhibits the RNase activity of Pru p 1.0101 but has no effect on Pru p 1.0201

• All three isoallergens interact with zeatin with comparable affinities (Kd values between 0.6 and 1.9 mM)

• NMR relaxation dispersion experiments show that the glycine-rich region is rigid and structurally homogeneous in solution

• Antibody binding epitopes appear to be distinct from the zeatin binding site, which is located in the protein's cavity

Recent research suggests that structural flexibility might represent a critical determinant that relates ligand binding of PR-10 allergens to immunologic reactions, with studies of hazelnut allergens (Cor a 1.04) showing correlation between antibody IgE binding and structural flexibility .

What factors influence antibody response in longitudinal studies of allergen exposure?

While not directly related to Pru p 1.01, other longitudinal studies of antibody responses provide valuable methodological insights:

Key influencing factors:

• Patient demographics (age, sex, genetic background)

• Clinical history and symptom profile

• Pre-existing comorbidities

• Occupational exposure

Longitudinal analysis approach:

• Sequential serological tests at defined time points

• Multiple FDA-approved immunoassay methods

• Correlation of antibody titers with clinical parameters

• Statistical analysis using appropriate methods for repeated measures

Study design considerations:

• Sample size and diversity considerations

• Time intervals between measurements

• Selection of appropriate controls

• Ethical considerations and informed consent

In one longitudinal study of COVID-19 patients, those with one or more comorbidities developed significantly better antibody titers at 14 months compared to those without comorbidities (p=0.033). Additionally, patients reporting specific symptoms (loss of smell and taste) developed significantly higher antibody titers, emphasizing the importance of correlating clinical features with antibody response patterns .

What are common challenges in developing specific antibodies against Pru p 1.01?

Researchers face several technical challenges when developing antibodies against Pru p 1.01:

Production challenges:

• Obtaining high-quality recombinant Pru p 1.01 for immunization

• Maintaining native-like conformation of immunogens

• Selecting optimal adjuvants to enhance immune response

• Balancing specificity and sensitivity in antibody selection

Cross-reactivity issues:

• High homology with other PR-10 proteins like Mal d 1 (apple allergen)

• Sequence similarity between different Pru p 1 isoallergens

• Need to distinguish between closely related protein family members

Methodological solutions:

• Careful selection of immunogenic epitopes unique to Pru p 1.01

• Rigorous screening of hybridoma cells using multiple techniques

• Extensive validation against related proteins (as shown in the specificity testing table)

• Application of different methods (ELISA, Western blot) to confirm specificity

How should researchers validate a new batch of Pru p 1.01 antibodies?

Validation of new antibody batches requires a systematic approach:

Essential validation steps:

• Purity assessment via SDS-PAGE and protein staining

• Specificity testing against recombinant isoallergens and natural extracts

• Sensitivity determination through titration series

• Functional testing in target applications (ELISA, Western blot, etc.)

• Batch-to-batch comparison with reference standards

Performance metrics to evaluate:

• Working concentration range

• Detection limits

• Recovery rates

• Intra- and inter-assay precision

• Cross-reactivity profiles

Application-specific validation:

• For ELISA: standard curve generation, LOD/LOQ determination

• For Western blot: testing against denatured and native protein forms

• For immunoprecipitation: verification of target pull-down efficiency

Research indicates that satisfactory antibody performance should include sensitivity in the ng/ml range for ELISA, specific recognition of the 17 kDa band for Western blot, and minimal cross-reactivity with non-target proteins like nPru p 3 or other PR-10 family members .