Allergen Bla g 4 Antibody, HRP conjugated

What is Allergen Bla g 4 and what is its biological significance?

Allergen Bla g 4 is a species-specific allergen produced by Blattella germanica (German cockroach) and expressed in a sex-specific manner in the reproductive system of adult male German cockroaches. It is associated with the spermatophore, transferred to the female during copulation, and developmentally regulated by juvenile hormones. Despite having low sequence identity with lipocalins (only 19-24%), Bla g 4 contains structurally conserved regions (SCR) characteristic of the lipocalin family . This allergen has been recognized as a significant contributor to cockroach allergy, which is a major cause of asthma in sensitive individuals. Its biological function appears to be related to reproductive processes in male cockroaches, while its allergenic properties stem from its ability to stimulate IgE production in humans, contributing to IgE-mediated allergic diseases .

What is the molecular structure of Bla g 4 and how does it relate to its allergenic properties?

Bla g 4 exhibits a structural configuration typical of lipocalins, characterized by a cup-shaped structure with 8 strands and an anti-parallel β-barrel shape . This structure is significant because it shares high structural homology with other allergenic lipocalins such as Rat n 1, Can f 1, Can f 2, Bos d 2, Bos d 5, Equ c 1, and Equ c 2, despite low sequence similarity . The protein has a molecular weight of approximately 24 kDa when expressed as a recombinant protein, somewhat larger than predicted from its amino acid sequence alone, which may be due to post-translational modifications or the contribution of tags in recombinant constructs .

The allergenicity of Bla g 4 appears to be related to specific IgE binding epitopes distributed throughout the molecule, with a major IgE-binding region located at amino acid residues 118 to 152, near the C-terminus of the protein . This epitope distribution pattern shows some similarities with the B-cell epitopes of other lipocalin allergens such as Bos d 5, suggesting conserved allergenic determinants within this protein family .

How can Allergen Bla g 4 Antibody, HRP conjugated be optimally used in ELISA assays?

For optimal use of Allergen Bla g 4 Antibody, HRP conjugated in ELISA assays, researchers should follow these methodological considerations:

• Coating Protocol: Dilute purified recombinant Bla g 4 to 10 μg/ml in coating buffer (0.1 M sodium carbonate, pH 9.6). Coat each well of a 96-well microplate with 100 μl of the recombinant protein and incubate at 4°C overnight, followed by washing with phosphate-buffered saline (pH 7.4) containing 0.05% Tween 20 (PBST) .

• Blocking Procedure: Add 200 μl of blocking solution (3% w/v skim milk in PBS) to each well and incubate at room temperature for 1 hour to prevent non-specific binding .

• Sample Preparation: Human serum samples should be diluted 1:4 in appropriate buffer before adding to wells and incubating for 2 hours .

• Antibody Application: For detection, add 50 μl of biotinylated anti-human IgE (1:1,000 dilution) followed by streptavidin-peroxidase (1:1,000 dilution), with appropriate washing steps between additions .

• Development and Analysis: Develop color using 3,3',5,5'-tetramethyl-benzidine (TMB) as substrate, incubate in the dark for 20 minutes, and stop the reaction with 0.5 M H₂SO₄. Read absorbance at appropriate wavelength depending on your substrate .

For experimental validation of results, always include positive and negative controls to ensure specificity and sensitivity of the assay.

What are the best practices for expression and purification of recombinant Bla g 4 for epitope studies?

Based on research findings, the following methodological approach is recommended for expression and purification of recombinant Bla g 4:

• Initial RNA Isolation: Extract total RNA from adult male B. germanica using established protocols (e.g., Trizol method) .

• cDNA Synthesis and Amplification: Perform RT-PCR using specific primers such as Bg4F (5'-GCAGTTTTGGCACTATGTGC-3') and Bg4R (5'-CTTAGTGACATGTGGAGTG-3'). PCR conditions: 35 cycles of 30 sec at 94°C, 30 sec at 50°C, and 1 min at 72°C, with initial denaturation at 95°C for 5 min and final extension at 72°C for 9 min .

• Cloning Strategy:

  • Clone the full-length cDNA into a suitable vector (e.g., pGEM-T Easy)

  • For expression studies, subclone into expression vector pET-28b using BamHI and XhoI restriction sites

  • Transform into E. coli BL21 (DE3) for protein expression

• Protein Expression:

  • For fragment analysis, divide Bla g 4 (EF202172) into 5 overlapping peptide fragments:

    • E1: amino acids 1-100

    • E2: amino acids 34-77

    • E3: amino acids 74-117

    • E4: amino acids 114-156

    • E5: amino acids 153-182

• Purification Strategy:

  • Express as N-terminal fusion proteins with 6 histidine residues

  • Note that fragments 1, 2, and 4 typically appear in insoluble fractions

  • Fragments 3 and 5 are generally found in soluble fractions

  • Purify using Ni-nitrilotriacetic acid-agarose chromatography

Typical protein yield is approximately 2.07 mg/L of culture. The purified recombinant full-length protein will migrate at approximately 24 kDa on SDS-PAGE, while peptide fragments may migrate at sizes different from their predicted molecular weights .

How should researchers analyze IgE binding epitopes of Bla g 4 using the antibody?

For comprehensive analysis of IgE binding epitopes of Bla g 4, researchers should implement the following methodological approach:

• Fragment-Based Epitope Mapping:

  • Generate overlapping recombinant peptide fragments spanning the entire Bla g 4 sequence

  • Express these fragments as described in question 2.2

  • Test each fragment for IgE reactivity using patient sera

• ELISA Protocol for Epitope Analysis:

  • Follow the ELISA protocol detailed in question 2.1

  • Test multiple sera from cockroach-allergic patients

  • Include appropriate controls (healthy human sera as negative controls)

• Data Analysis and Interpretation:

  • Consider result patterns across different patient sera to identify heterogeneity in IgE binding

  • Based on established research, expect potential epitope regions at amino acid sequences:

    • 34-73 (represented by fragment E2)

    • 78-113 (represented by fragment E3)

    • 118-152 (represented by fragment E4, which appears to contain a major IgE epitope)

• Confirmation Studies:

  • Consider site-directed mutagenesis of key residues within identified epitope regions

  • Previous studies have shown that mutations at Arg24, Arg26, and Lys75 can significantly reduce IgE binding in some patients

Research findings indicate heterogeneous IgE binding patterns among different patients, with the most consistent reactivity observed with fragment E4 (amino acids 114-156), suggesting this region contains a major IgE binding epitope. The heterogeneity in IgE responses to Bla g 4 reflects the polyclonal nature of immune responses to protein allergens .

What storage and handling protocols are recommended for Allergen Bla g 4 Antibody, HRP conjugated?

Based on product information, the following storage and handling guidelines are recommended:

• Storage Conditions: Upon receipt, store at -20°C or -80°C to maintain stability and activity. Avoid repeated freeze-thaw cycles as this can degrade the antibody and reduce its efficacy .

• Buffer Composition: The antibody is typically provided in a buffer containing preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4). This formulation helps maintain antibody integrity during storage .

• Working Solution Preparation: When preparing working dilutions, use freshly prepared buffers and maintain cold chain management to preserve antibody activity.

• Quality Control: Before experimental use, verify antibody activity using positive controls to ensure that storage conditions have not compromised functionality.

What factors might influence the variability in IgE binding to Bla g 4 across different patient populations?

Research on Bla g 4 has revealed several factors that contribute to variability in IgE binding across different patient populations:

• Protein Structure Modifications:

  • Minor modifications of the complete molecule through amino acid substitutions can affect 3-dimensional structure

  • Disruption of intramolecular disulfide bridges, which are characteristic of lipocalins, can interfere with IgE binding

• Multiple Isoforms:

  • The presence of multiple Bla g 4 isoforms may influence allergenicity

  • Similar to other major allergens (e.g., Birch pollen allergen Bet v 1 with 10 isoforms and mite allergen Der p 2 with 8 isoforms), Bla g 4 isoforms may differ in:

    • Extent of IgE-binding activity

    • T-cell activation potential

• Geographic and Population Variations:

  • Prevalence of Bla g 4 sensitization varies significantly between studies:

    • 40-60% reported by Arruda et al.

    • 17.4% (7/34) reported by Satinover et al.

    • 20.6% (7/34) reported by Slater et al.

    • 25% in Korean subjects per the cited study

  • These differences suggest regional variations in cockroach allergen exposure and immune responses

• Individual Immune Response Heterogeneity:

  • As demonstrated in epitope studies, different patients recognize different epitopes of Bla g 4

  • This heterogeneity reflects the polyclonal nature of immune responses to complex protein allergens

Understanding these factors is crucial for interpreting experimental results and developing targeted immunotherapeutic approaches.

How does Bla g 4 compare structurally and functionally with other lipocalin allergens?

Bla g 4 shares important structural and functional characteristics with other lipocalin allergens, though with notable distinctions:

• Sequence and Structural Comparison:

  • Sequence identity between Bla g 4 and other lipocalins is relatively low (19-24%)

  • Despite low sequence homology, Bla g 4 contains the structurally conserved regions (SCR) characteristic of lipocalins

  • Structural homology between Bla g 4 and other allergenic lipocalins (Rat n 1, Can f 1, Can f 2, Bos d 2, Bos d 5, Equ c 1, and Equ c 2) is high

• Epitope Conservation:

  • B-cell epitopes of Bla g 4 show some similarities with those of Bos d 5 (bovine β-lactoglobulin)

  • Major epitopes in Bos d 5 are located at residues 41-60, 102-124, and 149-162

  • This pattern is somewhat similar to Bla g 4's major epitope region at residues 118-152

• Functional Role:

  • Like other lipocalins, Bla g 4 likely functions as a carrier protein

  • Its expression in male reproductive tissue suggests a role in reproduction and mating

  • This is distinct from some other lipocalins involved in olfaction or pheromone transport

• Allergenicity Factors:

  • Intramolecular disulfide bridges, a characteristic of lipocalins, are important for maintaining allergenicity

  • Disruption of these bridges can significantly affect IgE binding, as seen in studies with mite allergens

This comparative analysis suggests that despite divergent evolutionary paths, allergenicity determinants may be conserved across the lipocalin family, which has implications for cross-reactivity and immunotherapy development.

What are the potential research applications of Bla g 4 Antibody in developing targeted immunotherapy?

Allergen Bla g 4 Antibody, HRP conjugated offers several promising applications in developing more specific and efficacious immunotherapy approaches:

• Epitope-Based Hypoallergen Development:

  • By identifying and characterizing IgE binding epitopes using Bla g 4 Antibody, researchers can design genetically modified recombinant allergens with reduced allergenic activity

  • Site-directed mutagenesis of key residues within identified epitopes (particularly in the 118-152 region) can create proteins that preserve T-cell epitope repertoire while reducing IgE binding capacity

• Immunotherapy Efficacy Assessment:

  • The antibody can be used to measure blocking antibody responses induced by immunotherapy

  • ELISA-based competition assays can determine if therapy-induced antibodies effectively block IgE binding to Bla g 4

• Population-Specific Therapy Design:

  • Given the heterogeneity in IgE binding across different patient populations, the antibody can help identify predominant epitopes in specific regional populations

  • This allows for customization of immunotherapy approaches based on regional sensitization patterns

• Cross-Reactivity Studies:

  • The antibody can be employed to investigate potential cross-reactivity between Bla g 4 and other lipocalin allergens

  • Such studies could inform broader immunotherapeutic strategies addressing multiple allergenic sources simultaneously

• Immunological Monitoring:

  • During clinical trials of cockroach allergy immunotherapy, the antibody can be used to monitor changes in antibody responses to specific epitopes

  • This provides valuable biomarkers for therapy efficacy and mechanism of action

Research using Bla g 4 Antibody has already demonstrated that "B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy" .

How might post-translational modifications affect Bla g 4 allergenicity and antibody binding?

Post-translational modifications (PTMs) can significantly influence Bla g 4 allergenicity and antibody binding through several mechanisms:

• Observed Effects on Protein Mobility:

  • Research has shown that recombinant Bla g 4 and its fragments often migrate at molecular weights higher than their predicted sizes on SDS-PAGE gels

  • This suggests the presence of PTMs that alter protein characteristics

• Sequence Diversity through Post-Transcriptional Modification:

  • Studies have found that sequence diversity of Bla g 4 can be produced by post-transcriptional modification

  • These modifications may create variant forms with different antigenic properties

• Impact on Epitope Accessibility:

  • PTMs can alter protein folding, potentially exposing or masking epitopes

  • This could explain some of the observed heterogeneity in IgE binding across patient populations

• Expression System Considerations:

  • Research has demonstrated that higher expression yields of Bla g 4 can be obtained in Pichia pastoris than in Escherichia coli

  • This suggests that yeast-specific PTMs may enhance protein stability or folding efficiency

• Research Strategy Implications:

  • When using Allergen Bla g 4 Antibody, researchers should consider how the source and preparation of Bla g 4 (natural vs. recombinant, prokaryotic vs. eukaryotic expression) might affect antibody binding

  • In some cases, multiple antibody preparations recognizing different epitopes may be needed to capture all relevant forms of the allergen

Future research should focus on characterizing specific PTMs in natural Bla g 4 and determining their impact on allergenicity and antibody recognition, which could inform more effective diagnostic and therapeutic approaches.

What methodological approaches can enhance the sensitivity and specificity of Bla g 4 detection in environmental samples?

For researchers working with environmental samples, enhancing the sensitivity and specificity of Bla g 4 detection requires sophisticated methodological approaches:

• Optimized Extraction Protocols:

  • Develop buffer systems that efficiently solubilize Bla g 4 from environmental dust samples

  • Consider sequential extraction methods to maximize allergen recovery while minimizing interferents

• Advanced Immunoassay Configurations:

  • Implement sandwich ELISA formats using two different epitope-specific antibodies

  • Consider using the HRP conjugated antibody as the detection antibody in a system where another antibody serves as the capture antibody

  • The protocol published for research applications involves:

    • Coating with antigen at 10 μg/ml in carbonate buffer (pH 9.6)

    • Blocking with 3% skim milk

    • Incubation with appropriate dilutions of test samples

    • Detection with biotinylated secondary antibody and streptavidin-peroxidase

    • Development with TMB substrate

• Pre-analytical Sample Processing:

  • Implement sample concentration techniques for low-abundance samples

  • Develop filtration or precipitation methods to remove inhibitory substances common in environmental samples

• Multiplex Detection Systems:

  • Develop bead-based multiplex assays that can simultaneously detect Bla g 4 along with other cockroach allergens

  • This approach increases throughput and provides a more comprehensive allergen profile

• Molecular Detection Alternatives:

  • Consider PCR-based methods targeting Bla g 4 DNA as a proxy for allergen presence

  • This may be particularly useful in samples where protein degradation is a concern

Each of these approaches should be validated using known quantities of purified recombinant Bla g 4 protein, ideally with the well-characterized epitope regions (E1-E5) serving as controls for antibody specificity .