Rabbit anti-Chicken Yolk Immunoglobulin Antibody;Biotin conjugated
Description
Introduction
Rabbit anti-Chicken Yolk Immunoglobulin (IgY) Antibody; Biotin conjugated is a specialized secondary antibody used in immunological assays to detect chicken-derived primary antibodies. Produced by immunizing rabbits with chicken IgY, this biotinylated reagent enables high-sensitivity detection through streptavidin-biotin binding systems, facilitating applications in Western blotting, immunohistochemistry, and ELISA .
Antibody Composition
Key Features
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Specificity: Binds to chicken IgY without cross-reactivity to non-immunoglobulin serum proteins .
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Avian vs. Mammalian Antibodies:
Common Uses
Signal Amplification
Biotin conjugation enables 3–5x greater signal amplification compared to non-conjugated secondary antibodies due to streptavidin’s high binding capacity .
Efficacy in Diagnostic Assays
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Biotin Interference: High biotin levels in egg yolk samples may require dilution adjustments in streptavidin-based assays .
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RF Interference Resistance: IgY shows no agglutination with rheumatoid factor (RF), unlike mammalian antibodies .
Therapeutic Potential
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Anti-Pathogen Activity: IgY antibodies inhibit bacterial adhesion (e.g., Helicobacter pylori) and viral entry (e.g., SARS-CoV-2) .
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Immunomodulation: Enhances T-helper 1 responses in Mycobacterium tuberculosis infections .
Limitations and Considerations
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Q&A
Basic Research Questions
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What is the molecular basis of chicken IgY and how does it differ from mammalian IgG?
Chicken yolk immunoglobulin Y (IgY) is the functional equivalent to mammalian IgG but differs in several important aspects. IgY is composed of two light chains and two heavy chains, with the heavy chains having a molecular mass of approximately 65,100 atomic mass units (amu), which is larger than those in IgG. The light chains in IgY have a molecular mass of about 18,700 amu, somewhat smaller than IgG light chains . The total molecular weight of IgY is approximately 167,000 amu.
Structurally, IgY contains an extra domain in its heavy chain that contributes to its enhanced stability compared to mammalian IgG. This structural difference gives IgY less steric flexibility than IgG . Functionally, IgY shares characteristics with both IgG and IgE, but importantly:
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Does not bind to Protein A or Protein G
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Does not bind to mammalian Fc receptors
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Does not activate the mammalian complement system
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Does not interact with rheumatoid factor or human anti-mouse IgG antibodies
These differences make IgY particularly valuable for certain immunodiagnostic applications where mammalian antibodies might produce background interference.
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What are the optimal storage conditions for biotin-conjugated Rabbit anti-Chicken IgY antibodies?
Proper storage is critical for maintaining antibody functionality. Based on manufacturer recommendations, biotin-conjugated Rabbit anti-Chicken IgY antibodies should be stored according to the following guidelines:
| Storage Phase | Temperature | Condition | Duration |
|---|---|---|---|
| Unopened | 4°C | Original container | Until expiration date |
| Opened, short-term | 4°C | Sealed container | Up to 1 month |
| Long-term storage | -20°C | With equal volume of glycerol (50% final) | Up to 12 months |
| Deep storage | -80°C | Aliquoted to avoid freeze-thaw cycles | Extended shelf life |
Most products are shipped with ice packs and should be stored immediately upon receipt . For lyophilized formats, reconstitution with the recommended volume of distilled water (typically to 1.3 mg/mL) is required before use . Importantly, repeated freeze-thaw cycles should be avoided as they may denature the antibody and reduce its binding activity .
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What applications are most suitable for biotin-conjugated Rabbit anti-Chicken IgY antibodies?
Biotin-conjugated Rabbit anti-Chicken IgY antibodies have demonstrated efficacy in multiple immunological techniques:
These antibodies are particularly valuable in detecting chicken IgY in research studying maternal antibody transfer to offspring, where they can be used to track egg yolk-derived antibodies during embryonic development and after hatching . The biotin conjugation provides amplification opportunities through streptavidin-based detection systems, enhancing sensitivity in various assays.
Intermediate Research Questions
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How does the stability of biotin-conjugated Rabbit anti-Chicken IgY compare to other secondary antibodies under variable conditions?
A comprehensive comparative study of IgY and rabbit IgG stability revealed significant differences that impact experimental design. The data shows:
| Condition | IgY Stability | Rabbit IgG Stability | Comparative Advantage |
|---|---|---|---|
| 30°C, 8h | Maintains >95% activity | Maintains >90% activity | Similar performance |
| 37°C, 8h | Maintains >90% activity | Maintains >85% activity | Similar performance |
| 60°C, 8h | Maintains ~70% activity | Drops to ~3.5% activity | IgY is ~20-fold more stable |
| pH 3-9 | Broad stability range | Narrower optimal range | IgY has better pH tolerance |
The extra domain present in the heavy chain of IgY plays a significant role in providing greater molecular stability compared to rabbit IgG under extreme conditions . This enhanced stability makes biotin-conjugated Rabbit anti-Chicken IgY particularly valuable for applications requiring harsh extraction conditions or prolonged incubation periods at elevated temperatures.
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What mechanisms govern IgY transport and how can they be studied using anti-IgY antibodies?
FcRY has been identified as a key receptor that regulates the transfer of IgY from maternal blood circulation into the egg yolk. Research using neutralizing antibodies against FcRY has provided critical insights into this process:
| Experimental Approach | Methodology | Findings |
|---|---|---|
| FcRY-specific antibody generation | Immunization of rabbits with purified secretory chicken FcRY | Produced antibodies with different neutralizing capacities |
| Neutralization assays | Intravenous injection of FcRY-specific antibody (1 mg/100g BW) with tracer IgY | Blocked IgY transfer from blood to yolk |
| Binding affinity studies | Surface plasmon resonance with immobilized FcRY | G365A mutant showed stronger binding to FcRY than wild-type IgY |
The G365A mutant of IgY-Fc with enhanced transport efficiency to egg yolk demonstrated higher binding affinity to FcRY than wild-type IgY. This was confirmed using surface plasmon resonance where FcRY (400 nM) was immobilized on sensor chips and exposed to different IgY-Fc variants (50-600 nM) at pH 5.5 .
Biotin-conjugated Rabbit anti-Chicken IgY antibodies serve as essential tools in these studies for detecting and quantifying the transferred IgY in both maternal circulation and egg yolk extracts.
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How can specificity be ensured when using Rabbit anti-Chicken IgY antibodies in complex immunological assays?
Ensuring specificity requires meticulous validation and control strategies:
| Validation Approach | Methodology | Purpose |
|---|---|---|
| Pre-adsorption controls | Incubation with excess target antigen before use | Confirms binding specificity |
| Cross-reactivity testing | Incubation with related avian immunoglobulins | Identifies potential false positives |
| Western blot analysis | Verification of specific band at ~180 kDa (IgY) | Confirms target recognition |
| Non-immunized control | Parallel testing with antibodies from non-immunized rabbits | Establishes background levels |
| Dilution linearity | Serial dilution of primary and secondary antibodies | Optimizes signal-to-noise ratio |
When developing immunoassays, it's crucial to recognize that while Rabbit anti-Chicken IgY antibodies primarily react with the heavy chains of chicken IgY and with light chains common to most chicken immunoglobulins, they may exhibit some cross-reactivity with immunoglobulins from other avian species . This cross-reactivity should be empirically determined for each specific application.
Advanced Research Questions
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What strategies can optimize the use of biotin-conjugated Rabbit anti-Chicken IgY in maternal antibody transfer studies?
Maternal antibody transfer studies require sophisticated experimental designs to track IgY movement. Advanced strategies include:
| Strategy | Methodology | Research Insights |
|---|---|---|
| In vivo tracking | Injection of DIG-labeled IgY with anti-FcRY antibodies | Revealed dose-dependent neutralization of IgY transport |
| Tissue-specific expression | Western blotting with anti-FcRY and anti-IgY antibodies | Identified FcRY expression in ovarian follicles |
| Immunofluorescence localization | Biotin-conjugated anti-rabbit IgG to detect anti-FcRY binding | Mapped receptor distribution in reproductive tissues |
| In ovo interventions | Injection of IgY variants into egg yolks | Demonstrated passage of maternal antibodies to offspring |
| Comparative mutant studies | G365A vs. Y363A mutants of IgY-Fc | Showed differential transport efficiency based on binding affinity |
In one pivotal study, fertilized eggs were injected with DIG-labeled IgY-Fc mutants (wild-type, Y363A, G365A) at 17 μg per egg, incubated until hatching, and then blood samples were collected to measure antibody transmission to offspring. The G365A mutant showed superior transfer efficiency, correlating with its stronger binding to FcRY (KD of 2.28 × 10−7 M compared to 3.33 × 10−7 M for wild-type) .
Biotin-conjugated Rabbit anti-Chicken IgY antibodies provide a versatile detection tool for these studies when coupled with streptavidin-based visualization systems.
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How can Rabbit anti-Chicken IgY antibodies be used to advance diagnostic and therapeutic applications of IgY technology?
IgY technology has emerging diagnostic and therapeutic applications that can be facilitated using well-characterized Rabbit anti-Chicken IgY antibodies:
| Application | Methodology | Potential Impact |
|---|---|---|
| Rotavirus diagnostics | Anti-RVA IgY detection using Rabbit anti-IgY in ELISA | Enhanced sensitivity pathogen detection |
| Passive immunotherapy | Monitoring therapeutic IgY levels in biological fluids | Optimized dosing regimens |
| Maternal immunity studies | Tracking IgY transfer efficiency to offspring | Improved vaccination strategies in poultry |
| Neutralization potency assays | Quantifying functional activity of therapeutic IgY | Quality control of immunotherapeutic preparations |
A study demonstrating the potential of anti-RVA IgY for prophylaxis and treatment of rotavirus-induced diarrhea utilized Rabbit anti-IgY antibodies conjugated with horseradish peroxidase (1:2000 dilution) to detect the binding of purified IgY to viral proteins . The neutralization ability of the purified IgY was confirmed by its capacity to block RVA activity in MA-104 cells.
The advantages of IgY-based therapeutics include their cost-effective production at scale, ethical advantages over mammalian antibody production systems, and enhanced stability under various conditions . Rabbit anti-Chicken IgY antibodies serve as essential tools for characterizing and validating these therapeutic preparations.
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What are the critical considerations for optimization when using biotin-conjugated Rabbit anti-Chicken IgY in multiplex immunoassays?
Multiplex immunoassays present unique challenges that require careful optimization when using biotin-conjugated Rabbit anti-Chicken IgY:
| Optimization Parameter | Methodology | Critical Considerations |
|---|---|---|
| Signal-to-noise optimization | Titration of primary and secondary antibodies | Typically 1:1000 dilution for biotin-conjugated anti-IgY with optimization needed for each assay format |
| Cross-reactivity management | Pre-absorption against common interferents | Particularly important with multiple avian species or when using complex biological matrices |
| Detection system selection | Comparison of different streptavidin conjugates | Quantum dot streptavidin conjugates may offer multiplexing advantages over traditional HRP or AP systems |
| Blocking optimization | Comparison of different blocking agents | BSA may be preferred over milk-based blockers due to potential glycoprotein interference |
| Sequential incubation strategies | Time-course experiments with varied incubation sequences | May reduce cross-talk between detection channels in multiplex formats |
Researchers should validate each antibody pair independently before combining them in multiplex formats. Importantly, when using biotin-conjugated antibodies in multiplex assays, endogenous biotin in samples may interfere with detection. This can be mitigated by using avidin pre-treatment steps to block endogenous biotin or by employing alternative detection systems for one or more of the analytes .
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How does the molecular stability of biotin-conjugated Rabbit anti-Chicken IgY impact experimental design across different research applications?
The enhanced molecular stability of IgY compared to mammalian IgG has significant implications for experimental design:
| Research Application | Impact of IgY Stability | Experimental Design Considerations |
|---|---|---|
| Field-based diagnostics | Extended shelf-life at ambient temperatures | Potential for deployment in resource-limited settings without cold chain |
| High-temperature extraction protocols | Maintained activity at 60°C | Enables more aggressive antigen retrieval methods in IHC applications |
| Long-term monitoring studies | Reduced degradation during storage | More consistent results across extended experimental timeframes |
| pH-challenging environments | Broader pH stability profile | Application in gastrointestinal pathogen studies with extreme pH variations |
The comparative stability data showing IgY is approximately twenty-fold more stable than rabbit IgG at 60°C for up to 8 hours suggests that biotin-conjugated Rabbit anti-Chicken IgY antibodies may retain functionality under conditions where other secondary antibodies would fail . This stability advantage can be particularly valuable in applications requiring harsh sample preparation techniques or when working with field samples in suboptimal conditions.
Additionally, researchers should consider that while the antibody itself may remain stable, the biotin conjugate might show different stability profiles, potentially requiring additional validation when pushing the boundaries of conventional protocols.
