IL-6 PAT1F10AT Antibody

What is the IL-6 PAT1F10AT Antibody and What are Its Key Characteristics?

The IL-6 PAT1F10AT antibody is a mouse-derived monoclonal antibody that specifically targets human interleukin-6 (IL-6). It belongs to the IgG1 subclass with kappa light chains and is formulated at a concentration of 1mg/ml in PBS (pH 7.4) with 10% glycerol and 0.02% sodium azide .

IL-6 is a pleiotropic cytokine that plays pivotal roles in immune regulation, inflammation, and the pathophysiology of various diseases including rheumatoid arthritis. The PAT1F10AT clone has been specifically developed to recognize human IL-6 with high specificity, making it valuable for researching IL-6-mediated signaling pathways and immune responses .

Key characteristics include:

• Catalogue number: ANT-540

• Type: Mouse Anti-Human monoclonal antibody

• Immunoglobulin subclass: IgG1 heavy chain and kappa light chain

• Recognition of linear epitopes on the IL-6 protein

What Experimental Applications is the IL-6 PAT1F10AT Antibody Validated For?

The IL-6 PAT1F10AT antibody has been validated for numerous research applications, similar to other high-quality IL-6 antibodies. Based on antibody characteristics and performance data from comparable IL-6 antibodies, this clone can be effectively utilized in:

• Western blotting for detecting denatured IL-6 proteins

• Enzyme-linked immunosorbent assay (ELISA) for quantitative IL-6 detection

• Immunoprecipitation to isolate IL-6 from complex biological samples

• Immunocytochemistry/Immunohistochemistry to visualize IL-6 localization in cells and tissues

• Flow cytometry for identifying IL-6-producing cells

Anti-IL-6 antibodies have demonstrated effectiveness in these applications, with particular utility in investigating IL-6 signaling in inflammatory conditions and autoimmune disorders .

What are the Recommended Storage and Handling Conditions?

For optimal retention of antibody activity and stability:

• Store the antibody at -20°C for long-term storage

• For frequent use, aliquot and store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles to prevent protein denaturation

• The formulation (1mg/ml containing PBS, pH 7.4, 10% glycerol and 0.02% sodium azide) helps maintain stability during storage

• When diluting for assays, use buffers compatible with the intended application

• Handle according to standard laboratory practices for proteins containing sodium azide

The presence of 10% glycerol in the formulation serves as a cryoprotectant, while sodium azide prevents microbial contamination during storage .

How Does IL-6 PAT1F10AT Compare to Other Commercial Anti-IL-6 Antibodies?

While direct comparative data between PAT1F10AT and other antibodies is limited in the provided search results, we can draw insights from comparison studies of similar anti-IL-6 antibodies:

Anti-IL-6 antibodies can vary significantly in their binding characteristics and functional properties. For example, humanized anti-IL-6 antibodies like HZ-0408b have shown superior binding affinity compared to Siltuximab, an FDA-approved chimeric anti-IL-6 mAb .

Comparative characteristics to consider:

When selecting between different anti-IL-6 antibodies, researchers should consider the specific experimental requirements, including species cross-reactivity, epitope specificity, and functional characteristics .

What Controls Should Be Used with IL-6 PAT1F10AT Antibody?

Proper experimental controls are essential for validating results obtained with the IL-6 PAT1F10AT antibody:

Positive Controls:

• Cell lines known to express IL-6 (e.g., stimulated peripheral blood mononuclear cells)

• Recombinant human IL-6 protein

• Tissues with confirmed IL-6 expression (e.g., inflamed synovial tissue)

Negative Controls:

• Isotype control antibody (mouse IgG1, kappa) at equivalent concentration

• Cell lines with confirmed absence of IL-6 expression

• IL-6 knockout or knockdown samples

• Secondary antibody-only controls to assess background

Blocking Controls:

• Pre-incubation of the antibody with recombinant IL-6 to confirm specificity

• Competitive binding assays with unlabeled antibody

These controls help establish specificity and validate experimental findings, particularly important when investigating complex signaling pathways .

How Can the IL-6 PAT1F10AT Antibody Be Optimized for Specific Applications?

Application-specific optimization strategies can significantly enhance experimental outcomes:

For Western Blotting:

• Titrate antibody concentrations (typical starting range: 0.1-1 μg/ml)

• Test both reducing and non-reducing conditions

• Optimize blocking solutions to minimize background

• Consider transfer methods appropriate for the molecular weight of IL-6 (~21-28 kDa, depending on glycosylation)

For ELISA Development:

• Perform checkerboard titrations to determine optimal coating concentration

• Compare direct vs. sandwich ELISA formats

• Assess different detection systems (colorimetric, chemiluminescent, fluorescent)

• Evaluate various blocking agents to improve signal-to-noise ratio

For Cell-Based Assays:

• Optimize fixation and permeabilization methods

• Determine appropriate antibody concentration and incubation conditions

• Consider cell type-specific variables that might affect IL-6 detection

Similar to experiments with other IL-6 antibodies, researchers should validate that PAT1F10AT recognizes the linear epitope of IL-6, which can be confirmed through techniques such as immunoblotting with heat-denatured IL-6 protein .

What Methodological Approaches Can Resolve Contradictory Results When Using IL-6 PAT1F10AT?

When faced with inconsistent or contradictory results:

Systematic Troubleshooting Approach:

• Antibody Validation: Confirm antibody activity using positive controls and standard curves with recombinant IL-6

• Sample Processing: Evaluate if sample preparation methods (lysis buffers, fixation protocols) affect epitope accessibility

• Interference Assessment: Test for potential interfering substances in samples (e.g., soluble IL-6 receptor)

• Cross-Reactivity Analysis: Examine potential cross-reactivity with related cytokines

• Experimental Variables: Standardize experimental conditions (temperature, incubation times, buffer compositions)

Advanced Resolution Strategies:

• Employ multiple detection methods to corroborate findings

• Use complementary antibodies targeting different IL-6 epitopes

• Implement genetic approaches (siRNA, CRISPR) to validate antibody specificity

• Consider kinetic analyses to understand temporal aspects of IL-6 signaling

Researchers investigating IL-6 signaling pathways should consider that both membrane-bound and soluble forms of IL-6 receptor exist, which can complicate interpretation of results .

How Can IL-6 PAT1F10AT Be Used to Investigate IL-6 Signaling Pathways?

IL-6 signaling involves complex pathways that can be investigated using targeted approaches:

STAT3 Signaling Analysis:
IL-6 primarily signals through the JAK-STAT pathway, particularly STAT3. The PAT1F10AT antibody can be used to:

• Block IL-6 binding to its receptor in cell culture experiments

• Evaluate downstream effects on STAT3 phosphorylation

• Assess changes in target gene expression

Similar to studies with other IL-6 antibodies, researchers can examine phosphorylated STAT3 levels following IL-6 stimulation with and without antibody neutralization .

Receptor Complex Studies:

• Investigate formation of IL-6/IL-6R/gp130 hexameric signaling complexes

• Examine classical signaling (via membrane-bound IL-6R) vs. trans-signaling (via soluble IL-6R)

• Assess receptor internalization and recycling dynamics

Functional Readouts:

• Measure acute phase protein production (e.g., SAA, CRP) in hepatic cells

• Assess B cell proliferation and antibody production

• Evaluate T cell differentiation and cytokine production

• Monitor effects on inflammatory mediator release

Studies with other anti-IL-6 antibodies have demonstrated their utility in inhibiting IL-6-stimulated serum amyloid A (SAA) secretion and IL-6-dependent cell proliferation, approaches that could be adapted for PAT1F10AT .

What Considerations Should Be Made When Using IL-6 PAT1F10AT for Inhibition Studies?

When designing inhibition experiments:

Dose-Response Relationships:

• Establish complete dose-response curves to determine IC50 values

• Compare with reference inhibitors (e.g., Siltuximab, Olokizumab) where possible

• Consider using multiple concentrations spanning at least 3 logs (e.g., 0.01-10 μg/ml)

Timing Considerations:

• Pre-incubation of antibody with IL-6 before adding to cells may enhance inhibition

• Evaluate both acute and chronic inhibition time courses

• Consider potential compensatory mechanisms that may emerge during extended inhibition

Readout Selection:

• Choose appropriate functional readouts based on cell type and research question

• For hepatocytes, SAA or CRP production serves as relevant endpoints

• For B cells, proliferation or immunoglobulin production can be measured

• For inflammatory models, cytokine production profiles provide valuable insights

Based on studies with other anti-IL-6 antibodies, expect dose-dependent inhibition of IL-6-stimulated responses, with complete inhibition potentially achievable at concentrations of approximately 10 μg/ml .

How Does Species Cross-Reactivity Affect Experimental Design with IL-6 PAT1F10AT?

The species specificity of PAT1F10AT has important implications for experimental design:

Cross-Reactivity Considerations:

• As a mouse anti-human antibody, PAT1F10AT is primarily designed to recognize human IL-6

• Cross-reactivity with IL-6 from other species should be empirically determined

• Limited cross-reactivity with mouse IL-6 would restrict its utility in murine models

Experimental Model Selection:

• Human cell lines and primary human cells are optimal test systems

• Humanized mouse models expressing human IL-6 may be appropriate

• Non-human primate models may be suitable if cross-reactivity is established

• Consider xenograft models where human IL-6-producing cells are implanted in immunodeficient mice

Immunogenicity Concerns for In Vivo Studies:

• The mouse origin of PAT1F10AT may trigger anti-mouse antibody responses in non-murine species

• This could limit long-term studies in certain animal models

• Consider using species-specific anti-IL-6 antibodies for prolonged in vivo studies

Similar anti-IL-6 antibodies have shown high species specificity and low cross-reactivity, which can be advantageous for specifically targeting human IL-6 but limiting for certain animal models .

What Methods Can Validate the Epitope Specificity of IL-6 PAT1F10AT?

Validating epitope specificity is crucial for understanding antibody function:

Epitope Mapping Approaches:

• Western blotting with denatured IL-6: Can determine if PAT1F10AT recognizes a linear or conformational epitope, similar to studies performed with HZ-0408b

• Peptide arrays: Overlapping peptides spanning the IL-6 sequence can pinpoint the specific binding region

• Competition assays: Competing with antibodies of known epitope specificity can provide indirect evidence of binding sites

• Hydrogen-deuterium exchange mass spectrometry: Offers detailed mapping of antibody-antigen interaction surfaces

• X-ray crystallography or cryo-EM: Provides structural definition of the antibody-antigen complex

Functional Epitope Validation:

• Test if PAT1F10AT blocks IL-6 binding to IL-6R using competitive binding assays

• Evaluate inhibition of IL-6/IL-6R/gp130 complex formation

• Compare neutralizing capacity with antibodies targeting different IL-6 epitopes

Studies with other anti-IL-6 antibodies have used immunoblot assays with heat-denatured recombinant IL-6 and competition ELISAs to characterize epitope recognition patterns .

How Can IL-6 PAT1F10AT Be Applied in Multi-Parameter Analysis of Inflammatory Responses?

Advanced multi-parameter approaches enhance the utility of IL-6 PAT1F10AT in complex experimental systems:

Multiplexed Analysis Strategies:

• Cytokine profiling: Combine with detection of other inflammatory mediators (TNF-α, IL-1β, IL-8) to assess comprehensive inflammatory signatures

• Phospho-flow cytometry: Simultaneously detect intracellular signaling events (p-STAT3, p-ERK, p-AKT) in conjunction with surface markers

• Mass cytometry (CyTOF): Incorporate PAT1F10AT into panels of 30+ parameters to characterize complex cellular responses

• Single-cell RNA-seq with protein detection: Correlate IL-6 protein levels with transcriptional profiles at single-cell resolution

Temporal and Spatial Analysis:

• Live-cell imaging: Use fluorescently labeled PAT1F10AT to track IL-6 dynamics in real-time

• Tissue microenvironment studies: Apply PAT1F10AT in multiplex immunohistochemistry to characterize IL-6 producing cells in tissue context

• Microfluidic systems: Investigate IL-6 production and signaling under controlled flow conditions

Systems Biology Integration:

• Incorporate IL-6 pathway data obtained using PAT1F10AT into computational models

• Integrate with omics datasets to understand IL-6's role in global regulatory networks

• Develop predictive models of therapeutic responses to IL-6 blockade

These integrated approaches provide a more comprehensive understanding of IL-6 biology in complex inflammatory conditions, similar to studies performed with other IL-6 targeting antibodies in rheumatoid arthritis models .