Rabbit anti-Human IgG Antibody

What is Rabbit anti-Human IgG antibody and how is it produced?

Rabbit anti-Human IgG antibodies are polyclonal antibodies generated by immunizing rabbits with purified human IgG. The process typically involves:

• Hyperimmunization of rabbits with human IgG antigens

• Collection of antisera from immunized rabbits

• Purification via affinity chromatography using human IgG covalently linked to agarose

• Optional conjugation to reporter molecules (e.g., HRP, biotin)

These antibodies are primarily used as secondary detection reagents in immunoassays to recognize human antibodies bound to target antigens .

What epitopes does Rabbit anti-Human IgG (H+L) recognize?

Rabbit anti-Human IgG (H+L) has dual specificity:

• Reacts with both heavy and light chains of human IgG

• Cross-reacts with the light chains of human IgM and IgA due to structural homology

This dual recognition pattern explains why these antibodies can detect multiple human immunoglobulin classes when the light chains are involved .

How does the specificity of Rabbit anti-Human IgG compare to antibodies from other species?

Rabbits produce antibodies with several advantages over rodent-derived antibodies:

The evolutionary distinction between rabbits (Lagomorpha) and rodents (Rodentia) contributes to the unique characteristics of rabbit antibodies, making them particularly valuable for certain applications .

What are the primary research applications for Rabbit anti-Human IgG antibodies?

Rabbit anti-Human IgG antibodies are versatile reagents validated for multiple applications:

The choice of specific format (unconjugated, HRP-conjugated, or biotinylated) depends on the experimental design and detection system .

How should Rabbit anti-Human IgG antibodies be used in ELISA assays?

For optimal ELISA performance with Rabbit anti-Human IgG antibodies:

• Capture ELISA format:

  • Coat plates with Rabbit anti-Human IgG at 1-10 μg/mL in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

  • Block with appropriate blocking buffer (BSA or serum-based)

  • Add human sample containing IgG

  • Detect with enzyme-conjugated antigen or another detection antibody

• Indirect ELISA format:

  • Coat plates with target antigen

  • Add human serum/antibody samples

  • Use HRP-conjugated Rabbit anti-Human IgG (typically at 1:1000-1:5000 dilution)

  • Develop with appropriate substrate

For quantitative assays, researchers should establish a standard curve using purified human IgG .

What considerations are important when using these antibodies in immunohistochemistry?

For successful immunohistochemistry applications:

• Antigen retrieval: Often necessary for formalin-fixed tissues

• Blocking endogenous peroxidase: Critical when using HRP-conjugated antibodies

• Dilution optimization: Typically 1:100-1:500 for most applications

• Minimizing background: Use antibodies with appropriate cross-adsorption (e.g., mouse-adsorbed versions for mouse tissues)

• Visualization systems: Biotin-conjugated versions work well with avidin-based detection systems

Rabbit anti-Human IgG antibodies have demonstrated superior sensitivity in IHC applications compared to mouse-derived antibodies in multiple comparative studies .

What are the different conjugate options available for Rabbit anti-Human IgG antibodies?

Various conjugated formats serve different experimental needs:

The choice depends on detection system requirements, sensitivity needs, and experimental design .

How critical is cross-adsorption for experimental applications?

Cross-adsorption significantly impacts experimental outcomes:

• Standard (non-adsorbed) antibodies: May react with immunoglobulins from multiple species

• Species-adsorbed versions: Minimal reactivity to specified species (e.g., mouse-adsorbed)

For experiments involving multiple species (e.g., human cells in mouse models, human antibodies detected on mouse tissues), mouse-adsorbed versions are essential to avoid false positive signals .

Research indicates that proper cross-adsorption can reduce background by up to 90% in complex multi-species experimental systems while maintaining target binding affinity .

What storage and handling conditions are recommended for maintaining antibody activity?

To preserve antibody functionality:

• Storage temperature: 2-8°C for short-term; aliquot and freeze at -20°C for long-term

• Avoid freeze-thaw cycles: Repeated freezing and thawing can denature antibodies

• Buffer considerations: Most formulations contain sodium azide (<0.1%) as preservative

• Dilution recommendations: Use fresh buffer systems for working dilutions

• Stability: Most preparations remain stable for at least 12 months when properly stored

Working dilutions should be prepared fresh and can typically be stored at 4°C for up to one week .

How can Rabbit anti-Human IgG antibodies be used in analyzing immune responses to infectious diseases?

These antibodies play crucial roles in infectious disease research:

• Serological monitoring: Detection of pathogen-specific human antibodies (IgG)

• Neutralization assay development: Assessment of functional antibody responses

• Epitope mapping: Characterization of human antibody binding sites

• Immune complex detection: Identification of antigen-antibody complexes in tissues

Recent applications include:

• COVID-19 research: Detection of SARS-CoV-2-specific antibodies, including analysis of how biliverdin can modulate spike protein recognition by human antibodies

• Development of synthetic rabbit-human antibody conjugates as positive controls for hepatitis E virus (HEV) IgM assays

• Analysis of neutralizing antibody responses against viral pathogens

These applications demonstrate the versatility of these reagents in infectious disease research settings .

What are the considerations when using these antibodies in multiplexed assay formats?

For multiplexed detection systems:

• Cross-reactivity assessment: Validate specificity against all targets in the multiplex panel

• Signal optimization: Balance signal intensities across different targets

• Conjugate selection: Choose compatible fluorophores or enzyme systems to avoid spectral overlap

• Background minimization: Use thoroughly cross-adsorbed antibodies

• Blocking optimization: Test different blocking reagents to minimize non-specific binding

Researchers should perform comprehensive validation studies including single-target controls alongside multiplexed samples to ensure specificity and sensitivity .

How have technological advances impacted the production and use of Rabbit anti-Human IgG antibodies?

Recent technological developments include:

• Rabbit hybridoma technology: Generation of rabbit monoclonal antibodies with high specificity and affinity

• Transgenic rabbits: Production of fully human antibodies in rabbits for therapeutic applications

• Recombinant rabbit monoclonal antibody (RMAb) platforms: Monitoring specificity and neutralizing activities of antibodies

• Chimeric rabbit/human Fab libraries: Enhanced diversity and selection capabilities

• High-throughput DNA sequencing: Analysis of rabbit antibody repertoires and deconvolution of monoclonal antibodies

These advances have expanded the utility of rabbit-derived antibodies, particularly in therapeutic and diagnostic applications requiring high specificity and affinity .

What are common causes of background or non-specific binding when using Rabbit anti-Human IgG antibodies?

Background issues typically arise from:

• Cross-reactivity with endogenous Fc receptors: Block with appropriate Fc block reagents

• Insufficient blocking: Optimize blocking buffer composition and incubation time

• Endogenous peroxidase activity: Use appropriate quenching steps for HRP-conjugated antibodies

• Antibody concentration: Excessive antibody concentrations can increase non-specific binding

• Sample-specific factors: Human samples may contain rheumatoid factor or heterophilic antibodies

Troubleshooting approaches include:

• Titration of antibody concentration

• Testing different blocking agents (BSA, casein, normal serum)

• Including detergents (0.05-0.1% Tween-20) in wash buffers

• Using species-adsorbed antibody variants .

How can researchers validate the specificity of Rabbit anti-Human IgG in complex experimental systems?

Validation strategies include:

• Isotype controls: Use purified rabbit IgG as negative control

• Competitive inhibition: Pre-incubation with soluble human IgG should reduce specific binding

• Western blot analysis: Confirm recognition of human IgG heavy and light chains

• Cross-reactivity testing: Evaluate binding to IgG from other species

• Human IgG-depleted samples: Negative controls should show minimal reactivity

For complete validation, researchers should include appropriate positive and negative controls and assess potential cross-reactivity with all components in their experimental system .

What approaches can optimize signal-to-noise ratio in challenging samples?

For difficult samples with high background:

• Antibody titration: Determine optimal concentration that maximizes specific signal while minimizing background

• Buffer optimization: Adjust salt concentration and pH to improve specificity

• Cross-adsorption: Use highly cross-adsorbed antibodies in multi-species systems

• Blocking optimization: Test different blocking agents (e.g., BSA, casein, normal serum, commercial blockers)

• Alternative detection systems: Signal amplification methods (e.g., tyramide signal amplification) can improve sensitivity without increasing background

For particularly challenging applications, consider:

• Two-step detection systems with biotinylated primary and streptavidin-conjugated reporter

• Using F(ab')₂ fragments instead of whole IgG to reduce Fc-mediated interactions

• Including carrier proteins or detergents in dilution buffers .