IL-6 PAT1H6AT Antibody

What is IL-6 and what are its primary functions in the immune system?

IL-6 is a pleiotropic cytokine that functions as both a pro-inflammatory and anti-inflammatory mediator. It was initially identified as a B cell growth factor and inducer of plasma cell differentiation. IL-6 is produced by multiple cell types, including antigen-presenting cells, T cells, and macrophages, as a phosphorylated and variably glycosylated molecule .

IL-6 plays essential roles in:

• Final differentiation of B-cells into immunoglobulin-secreting cells

• Lymphocyte and monocyte differentiation

• Antibody production and class switching in vivo

• Directing the transition from innate to acquired immunity

• Hematopoiesis, bone metabolism, and cancer progression

IL-6 interacts with the IL-6 receptor and glycoprotein 130, activating downstream signaling pathways that mediate its diverse biological effects .

How do I select the appropriate IL-6 antibody for my experiment?

When selecting an IL-6 antibody, consider the following factors:

For example, antibody 66146-1-Ig has been validated for Western blot (1:1000-1:6000 dilution), Immunofluorescence (1:400-1:1600), and other applications with human samples . Always check if the antibody has been validated in cells or tissues similar to your experimental system.

What controls should I include when using IL-6 antibodies?

Proper controls are essential for interpreting results with IL-6 antibodies:

• Positive controls: Use cell lines known to express IL-6, such as U-87 MG cells, Jurkat cells, or LPS-stimulated HUVEC cells

• Negative controls: Include IL-6 knockout/knockdown samples or isotype control antibodies

• Stimulation controls: Compare unstimulated vs. stimulated conditions (e.g., LPS treatment increases IL-6 expression)

• Blocking peptide controls: Use for validating antibody specificity

• Secondary antibody-only controls: To assess non-specific binding

For ELISA applications, standard curves using recombinant IL-6 protein are essential for quantification .

How does IL-6 mediate antibody production, and what implications does this have for experimental design?

Recent research has revealed that IL-6 does not directly promote antibody production by B cells as previously thought. Instead, IL-6 acts through an indirect pathway:

• IL-6 induces IL-21 production by naïve and memory CD4+ T cells upon T cell receptor stimulation

• IL-21 produced by these CD4+ T cells then acts directly on B cells to promote antibody production

• This IL-21-dependent pathway is essential for IL-6-mediated enhancement of antibody responses

Evidence supporting this mechanism includes:

• IL-6 receptor (IL-6Rα) expression is minimal on resting B cells but substantial on CD4+ T cells

• IL-6 fails to induce IgG1 production when B cells are cultured with IL-21-deficient CD4+ T cells

• B cells from IL-21R-deficient mice do not produce IgG1 in response to IL-6 stimulation despite co-culture with capable CD4+ T cells

For experimental design, researchers should:

• Consider both IL-6 and IL-21 signaling pathways when studying antibody responses

• Include measurements of IL-21 when examining IL-6 effects on B cells

• Use IL-21 or IL-21R knockout models to confirm the mechanism

• Assess CD4+ T cell involvement in IL-6-mediated B cell responses

What methodological approaches are recommended for detecting intracellular versus secreted IL-6?

Detection of intracellular and secreted IL-6 requires different methodological approaches:

For intracellular IL-6 detection:

• Immunofluorescence (IF): Optimal at 1:400-1:1600 dilution for antibody 66146-1-Ig

• Flow cytometry: Requires cell permeabilization

• Western blot: Can detect the 24 kDa IL-6 protein in cell lysates

• Consider using Brefeldin A to block protein secretion and enhance intracellular detection

For secreted IL-6 detection:

• ELISA: Use matched antibody pairs such as MAB2063 for detection

• Commercially available DuoSet or Quantikine ELISA kits (e.g., DY206, D6050)

• Multiplex bead-based assays for simultaneous detection of multiple cytokines

When studying stimulated cells, the timing of sample collection is critical as IL-6 secretion kinetics vary by cell type and stimulus.

How can I use IL-6 antibodies to study the relationship between IL-6 and viral infections?

IL-6 plays crucial roles in antiviral immune responses, particularly against influenza. Research strategies using IL-6 antibodies include:

• Monitoring IL-6 production during infection:

  • Use IHC or IF to identify IL-6-producing cells in infected tissues

  • Quantify IL-6 levels in serum or bronchoalveolar lavage fluid by ELISA

• Investigating antibody responses:

  • IL-6 administration during influenza immunization enhances virus-specific IgG1 production (but not IgM)

  • This enhancement is dependent on IL-21, as demonstrated in IL-21-deficient mice

• Protection studies:

  • IL-6-deficient mice show increased susceptibility to influenza virus infection

  • These mice cannot recover from weight loss caused by acute viral infection, indicating IL-6 is essential for protection

• Mechanistic studies:

  • Combine IL-6 detection with IL-21 measurement to examine the sequential cytokine pathway

  • Use neutralizing IL-6 antibodies to block IL-6 signaling and assess effects on viral clearance

Research has shown that while influenza-specific IgM levels are comparable between wild-type and IL-21-deficient mice, IgG1 and IgG2c levels are severely reduced in IL-21-deficient mice, highlighting the importance of this IL-6/IL-21 axis in antiviral antibody responses .

What are the critical factors for optimizing Western blot detection of IL-6?

Optimizing Western blot detection of IL-6 requires attention to several factors:

Sample preparation:

• IL-6 has a molecular weight of 24 kDa but may appear at different sizes due to post-translational modifications

• Consider using positive control samples such as U-87 MG cells, Jurkat cells, or HUVEC cells

• IL-6 is a secreted protein; culture supernatants may be concentrated for detection

Protocol optimization:

• Recommended antibody dilution: 1:1000-1:6000 for antibody 66146-1-Ig

• Blocking: 5% non-fat milk or BSA in TBST

• Secondary antibody selection based on primary antibody species and isotype (e.g., Mouse IgG2b for 66146-1-Ig)

• Enhanced chemiluminescence detection systems for optimal sensitivity

Troubleshooting:

• Multiple bands may indicate different glycosylation states or degradation products

• Weak signal may require longer exposure times or higher antibody concentration

• Non-specific binding can be reduced with more stringent washing or different blocking reagents

How can IL-6 antibodies be used to investigate IL-6's role as a potential coadjuvant for vaccines?

Research has demonstrated that IL-6 can enhance humoral immunity, suggesting potential applications as a vaccine coadjuvant. Experimental approaches using IL-6 antibodies include:

• Vaccination studies:

  • Administer IL-6 with inactive virus/antigen and measure enhanced virus-specific antibody production

  • Compare different immunization protocols with and without IL-6 supplementation

  • Use ELISA with anti-IL-6 antibodies to monitor IL-6 levels throughout immune response

• Mechanistic investigation:

  • Track IL-21 upregulation in CD4+ T cells following IL-6 administration

  • Confirm IL-21 dependency using IL-21-deficient mice

  • Monitor class switching from IgM to other isotypes (IgG1, IgG2a, IgG3)

• Antibody response assessment:

  • Measure isotype-specific antibody responses (IL-6 enhances IgG1 but not IgM)

  • Evaluate antibody affinity and neutralizing capacity

  • Assess long-term antibody persistence

What are the optimal storage conditions for IL-6 antibodies?

For maximum stability and performance, IL-6 antibodies should be stored according to manufacturer recommendations:

• Store at -20°C for long-term stability

• Antibodies are typically stable for one year after shipment when properly stored

• Storage buffers usually contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations (e.g., 20μl sizes) may contain 0.1% BSA for additional stability

Avoid repeated freeze-thaw cycles, which can compromise antibody performance. Always return antibodies to recommended storage temperatures promptly after use.

How can I troubleshoot non-specific binding issues with IL-6 antibodies?

Non-specific binding can complicate interpretation of results when using IL-6 antibodies. Consider these troubleshooting approaches:

For Western blot applications:

• Increase blocking time/concentration (5-10% blocking agent)

• Use alternative blocking agents (milk vs. BSA)

• Optimize antibody dilution (test range from 1:1000-1:6000)

• Increase washing duration and frequency

• Include detergents (0.1-0.3% Tween-20) in washing buffers

• Use highly purified antibodies (e.g., Protein A purified)

For immunofluorescence/IHC:

• Optimize fixation methods

• Include appropriate serum block based on secondary antibody species

• Test different antibody dilutions (1:400-1:1600 range)

• Use appropriate controls including isotype controls and IL-6 knockout/knockdown samples

• Consider alternative detection systems or amplification methods

What is the best approach for validating antibody specificity for IL-6?

Validating antibody specificity is critical for reliable results. Comprehensive validation approaches include:

• Genetic models:

  • Test antibody in IL-6 knockout or knockdown samples

  • Compare siRNA or shRNA treated vs. non-treated cells

• Expression modulation:

  • Compare IL-6 detection in LPS-stimulated vs. unstimulated cells

  • Use Brefeldin A treatment to enhance intracellular accumulation for IF detection

• Orthogonal methods:

  • Confirm protein detection with multiple antibodies targeting different epitopes

  • Compare protein expression with mRNA levels (RT-PCR, RNA-seq)

  • Use recombinant IL-6 protein as positive control

• Mass spectrometry:

  • Confirm identity of detected band from immunoprecipitation

• Blocking experiments:

  • Pre-incubate antibody with recombinant IL-6 before application

Proper validation ensures reliable results and prevents misinterpretation of experimental data.