EBI3 Antibody

Basic Research Questions

• What is EBI3 and what are its primary functions in immunological research?

  EBI3 (Epstein-Barr Virus Induced Gene 3) is a 34 kDa soluble glycoprotein first identified in B cells following Epstein-Barr virus infection. It functions as a critical subunit in multiple heterodimeric cytokines of the IL-12 family. EBI3 can be expressed and secreted as monomers or homodimers, and was first identified in virally-infected B cells . Functionally, EBI3 demonstrates considerable versatility in immune regulation, contributing to both pro-inflammatory and anti-inflammatory responses depending on its heterodimerization partner. The protein is predominantly expressed in spleen, tonsils, and reaches elevated levels in full-term placenta, where it may play a role in immune tolerance during pregnancy .

• Which cytokines contain EBI3 as a constituent subunit?

  EBI3 serves as a key subunit in multiple heterodimeric cytokines:

  Cytokine	Composition	Primary Function
  IL-27	EBI3 + p28 (IL-30)	Balances pro-inflammatory and anti-inflammatory effects; promotes Th1 commitment through T-bet induction
  IL-35	EBI3 + p35 (IL-12 subunit)	Immunoregulatory; suppresses effector T cells; induces regulatory T cell phenotype
  IL-39	EBI3 + p19 (IL-23 subunit)	Implicated in inflammatory responses
  p40-EBI3	EBI3 + p40	Novel anti-inflammatory cytokine involved in suppressing immune responses

  EBI3 was first found to heterodimerize with p28 (IL-30) to form IL-27, which is secreted by antigen-presenting cells in response to pro-inflammatory stimuli . More recently, EBI3 was also found to dimerize with the IL-12 subunit, p35 to form IL-35 .

• What are the common applications for EBI3 antibodies in immunological research?

  EBI3 antibodies support multiple experimental applications:

  • Western blotting (WB): For detection of EBI3 protein in cell or tissue lysates

  • Immunoprecipitation (IP): To isolate EBI3 or its complexes from biological samples

  • Immunofluorescence (IF): For visualization of EBI3 in cells or tissues

  • Flow cytometry: For detection of intracellular EBI3

  • ELISA: For quantification of EBI3 in solution or as part of cytokine complexes

  • Functional blocking: To investigate EBI3's role in biological processes through inhibition

  The specific application determines the optimal antibody format and clone selection. For example, the ebic6 monoclonal antibody has been reported for use in intracellular staining followed by flow cytometric analysis , while the DNT27 monoclonal has been tested for western blot applications .

• What should I consider when selecting between monoclonal and polyclonal EBI3 antibodies?

  Selection criteria should be based on your specific experimental requirements:

  Monoclonal antibodies (e.g., ebic6, DNT27, G-4):

  • Offer high specificity for a single epitope

  • Provide consistent lot-to-lot reproducibility

  • Ideal for specific applications like flow cytometry where conformational epitopes are preserved

  • Available in various conjugated forms (PE, FITC, APC)

  • Particularly useful when discrimination between species is important (e.g., Clone A15058A does not react with mouse EBI3)

  Polyclonal antibodies (e.g., ab83896):

  • Recognize multiple epitopes on the target protein

  • May offer higher sensitivity for applications like western blotting

  • Useful when protein conformation might be altered (denatured proteins in WB)

  • May show greater batch-to-batch variation

  For critical experiments, validation with multiple antibody clones is recommended to confirm findings.

Advanced Research Questions

• How can I distinguish between free EBI3 and its heterodimeric forms (IL-27, IL-35) in experimental systems?

  Distinguishing between free EBI3 and its heterodimeric forms requires strategic approaches:

  Co-immunoprecipitation analysis:

  • Use EBI3-specific antibodies for immunoprecipitation followed by immunoblotting with antibodies against potential partner subunits (p28, p35)

  • As demonstrated in research, when EBI3 was immunoprecipitated with a specific mAb, co-immunoprecipitation of IL-6 could be detected in culture media containing both proteins

  Sequential immunoprecipitation:

  • Deplete samples of specific complexes using antibodies against partner subunits

  • Quantify remaining EBI3 to determine proportion in heterodimeric forms

  Specialized ELISA approaches:

  • Develop sandwich ELISAs using capture antibodies against partner subunits and detection antibodies against EBI3

  • This approach was used to detect IL-39 using Mouse Anti-Human IL‑23 p19 Monoclonal Antibody for capture and Mouse Anti-Human EBI3 Monoclonal Antibody for detection

  Size exclusion chromatography:

  • Separate protein complexes based on size before immunodetection

  • Identify fractions containing EBI3 alone (~34 kDa) versus heterodimeric complexes (~50-70 kDa)

• What are the optimal protocols for detecting intracellular versus secreted EBI3?

  For intracellular EBI3 detection:

  • Flow cytometry: Use cell permeabilization protocols optimized for cytokine detection

    • Fix cells with 4% paraformaldehyde

    • Permeabilize with 0.1-0.5% saponin or commercial permeabilization buffers

    • Incubate with fluorophore-conjugated anti-EBI3 antibodies like ebic6-PE or ebic6-APC

  • Immunofluorescence: Fix cells with paraformaldehyde, permeabilize with Triton X-100, and stain with anti-EBI3 antibodies followed by appropriate secondary antibodies

  For secreted EBI3 detection:

  • ELISA: Optimize sample collection by adding protease inhibitors to culture supernatants

  • Concentrated supernatants using Amicon Ultra-15 centrifugal filters improve detection sensitivity

  • Western blotting: Consider concentration steps for dilute samples

  Important considerations:

  • EBI3 can associate with calnexin in the endoplasmic reticulum, indicating that its secretion might depend on association with a second subunit

  • When coexpressed with p35 or p28, EBI3 secretion is mutually facilitated

  • Timing is critical: EBI3 secretion patterns vary with cell activation status

• How does EBI3 function in tumor microenvironments, and how can antibodies help investigate this relationship?

  EBI3 plays complex roles in tumor biology that can be investigated using various antibody-based approaches:

  Tumor-promoting mechanisms:

  • EBI3 expression is elevated in Hodgkin and Reed-Sternberg cells, suggesting a role in evading anti-tumor immunity

  • Through IL-35 formation, EBI3 may suppress anti-tumor immune responses by expanding regulatory T cells and inhibiting effector T cell function

  Investigative approaches:

  • Immunohistochemistry: Use anti-EBI3 antibodies to assess expression patterns in tumor tissues

    • The polyclonal antibody ab83896 has been validated for IHC-P applications in human tissues

  • Flow cytometry: Characterize EBI3 expression in tumor-infiltrating immune cells

  • EBI3 blocking experiments: EBI3 blocking peptides have been shown to promote antitumor cytotoxic T lymphocyte responses by inducing Granzyme B, IFN-γ production and proliferation of tumor-infiltrating lymphocytes

  Mechanistic findings:

  • EBI3 blocking leads to reduced tumor growth in vivo in colorectal cancer models

  • EBI3 associates with STAT3 signaling in tumor cells, with blocking experiments showing decreased gp130 and p-STAT3 expression

  • These findings suggest EBI3 may assist tumor immune escape through bidirectional STAT3 signaling pathway manipulation

• What are the challenges in studying EBI3 due to its participation in multiple cytokine complexes?

  Researchers face several methodological challenges when studying EBI3:

  Attribution of biological effects:

  • Effects observed after EBI3 manipulation could be due to alterations in IL-27, IL-35, IL-39, or even IL-6 trans-signaling

  • EBI3 can activate STAT3 and induce proliferation of IL-6-dependent cell lines by mediating IL-6 trans-signaling

  Functional redundancy:

  • Knockout studies may reveal limited phenotypes due to compensatory mechanisms

  • EBI3-deficient mice show complex phenotypes with diminished T helper type 1 and increased T helper type 2 responses

  Contextual expression:

  • EBI3 interaction partners vary by cell type and activation state

  • Tissue-specific effects require careful experimental design

  Technical solutions:

  • Use partner-specific knockouts in parallel with EBI3 knockouts

  • Develop antibodies that specifically block EBI3 interaction with individual partners

  • Employ proximity ligation assays to visualize specific heterodimeric complexes in situ

  • Use recombinant fusion proteins (like EBI3-IL-6) to distinguish effects of specific complexes

• What are the best approaches for co-immunoprecipitation experiments to study EBI3 interactions?

  Optimized co-immunoprecipitation protocols for EBI3 studies include:

  For cell lysate co-IP:

  • Lyse cells in buffer containing 1% NP-40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and protease inhibitors

  • Pre-clear lysates with protein A/G beads

  • Immunoprecipitate using EBI3-specific antibodies (1-5 μg per sample)

  • For agarose-conjugated antibodies, use EBI3 Antibody (G-4) AC

  • When studying tissue samples (like placenta), optimize lysis conditions (as described in source )

  For secreted protein co-IP:

  • Concentrate culture supernatants using centrifugal filters

  • For His-tagged proteins, use His-select® HF nickel affinity gel

  • In studies of EBI3-IL-6 interaction, biotinylated proteins were detected after elution using streptavidin-HRP

  Alternative approaches:

  • For multiple epitope detection, sequential immunoprecipitation can help isolate specific heterodimeric complexes

  • Surface plasmon resonance (SPR) provides quantitative binding data for EBI3 interaction studies

  Critical controls:

  • Include immunoprecipitation with isotype-matched control antibodies

  • When studying coexpressed proteins, include single-transfectant controls

• How can I optimize flow cytometry protocols for EBI3 detection in different cell populations?

  Flow cytometry optimization for EBI3 detection requires attention to several parameters:

  Cell preparation and fixation:

  • For intracellular staining, fix cells with 4% paraformaldehyde for 10-15 minutes

  • Use saponin-based permeabilization buffers optimized for cytokine staining

  Antibody selection and titration:

  • For human EBI3: Use ebic6 monoclonal antibody in fluorophore-conjugated formats (PE, APC)

  • For mouse EBI3: Use DNT27 clone, noting that some clones (like A15058A) do not cross-react with mouse EBI3

  • Titrate antibodies to determine optimal concentration (typically 0.5-5 μg/ml)

  Multiplexed staining strategies:

  • Combine EBI3 staining with surface markers to identify specific cell populations

  • Include intracellular staining for partner molecules (p28, p35) to assess co-expression

  • For studying activation status, include phospho-STAT3 (Tyr-705) staining

  Analysis considerations:

  • Use appropriate FMO (fluorescence minus one) controls for accurate gating

  • Consider time-course experiments as EBI3 expression may be transient

  • For T cell subsets analysis, combine with lineage markers (CD4, CD8) and functional markers (FOXP3 for Tregs)

  Exemplary protocol:
  As described in source , cells can be fixed, permeabilized, and stained with FITC-labeled anti-phospho-STAT3 (Tyr-705) and APC-labeled anti-CD4 mAbs to assess both signaling activity and cell identity.

• What approaches can be used to investigate the functional roles of EBI3 in disease models?

  Multiple strategies can elucidate EBI3's roles in disease contexts:

  Blocking antibodies and peptides:

  • EBI3 blocking peptides have demonstrated effects on antitumor immune responses

  • Measure outcomes such as cytotoxic T lymphocyte responses, cytokine production, and cell proliferation

  Genetic models:

  • EBI3-deficient mice show altered immune responses, with diminished Th1 and increased Th2-type airway inflammatory responses

  • These models reveal that EBI3 may play an inhibitory role in allergic asthma development

  Ex vivo functional assays:

  • Culture immune cells from disease models with EBI3 blocking agents

  • Measure functional outcomes like proliferation, cytokine production, and cytotoxicity

  In vivo therapeutic approaches:

  • Inject EBI3-targeting antibodies or blocking peptides into disease models

  • Assess tumor growth in xenograft models treated with EBI3 blocking peptides

  • Evaluate immune infiltration and activation status in tissues

  Important caution:
  Recent research indicates that EBI3 can promote pro-inflammatory IL-6 functions by mediating trans-signaling . This suggests that therapeutic use of EBI3 may require co-administration of soluble gp130 to prevent side effects associated with IL-6 trans-signaling .

• How do I interpret complex results when studying EBI3 in contexts where multiple binding partners may be present?

  Interpreting EBI3 results requires careful consideration of potential binding partners and context-dependent effects:

  Analytical framework:

  • Assume heterogeneity in EBI3 complexes within biological samples

  • Consider that effects may be due to one or multiple EBI3-containing cytokines

  • Evaluate observed phenotypes against known effects of IL-27, IL-35, and IL-6 trans-signaling

  Validation approaches:

  • Use partner-specific blocking antibodies to dissect contributions of individual complexes

  • Employ recombinant cytokines (IL-27, IL-35) as reference points

  • Utilize cells lacking specific receptor components to distinguish signaling pathways

  Contextual considerations:

  • Cell type specificity: Different cells express different receptors for EBI3-containing cytokines

  • Temporal dynamics: Expression of EBI3 and partner molecules varies with activation state

  • Environmental factors: Inflammation status can alter the balance of cytokine complex formation

  Illustrative example:
  In studies of B9 plasmacytoma cells, researchers found that EBI3-induced proliferation could be blocked by either anti-gp130 or anti-IL-6 mAbs, suggesting that the effect was mediated through IL-6 trans-signaling rather than through IL-27 or IL-35 pathways .