IL37 Antibody

Basic Research Questions and Methodology

• What is IL-37 and why are antibodies against it important for research?

  IL-37 is a member of the interleukin 1 cytokine family that functions as a natural inhibitor of immune responses. It suppresses innate inflammatory and immune responses, exhibiting anti-inflammatory characteristics resembling those of IL-1R8 . IL-37 antibodies are critical research tools that enable detection, quantification, and functional analysis of IL-37 in biological samples. These antibodies help researchers study IL-37's role in various disease models, inflammatory processes, and its potential as a therapeutic target . The human version of IL-37 has a canonical amino acid length of 218 residues and a protein mass of 24.1 kilodaltons .

• What are the different isoforms of IL-37 and how do they differ?

  IL-37 comprises five different isoforms, named as IL-37a–e:

  Isoform	Key Characteristics	Functional Status
  IL-37a	Contains exons 1, 2, 4, 5, and 6	Functional
  IL-37b	Contains exons 1, 2, 4, 5, and 6 (most complete)	Most studied, functional
  IL-37c	Lacks one or more of exons 4, 5, and 6	Likely non-functional
  IL-37d	Contains exons 1, 4, 5, and 6	Functional
  IL-37e	Lacks one or more of exons 4, 5, and 6	Likely non-functional

  IL-37 isoforms a, b, and d share exons 4, 5, and 6, which encode functional proteins involved in the formation of the beta-fold barrel structure essential for the extracellular functional activity . The IL-37 isoforms c and e lack one or more of these exons, thus may encode non-functional proteins .

• What are the common applications of IL-37 antibodies in research?

  IL-37 antibodies are used in multiple research applications:

  • Western Blotting (WB): Detects IL-37 protein expression levels in cell or tissue lysates, typically revealing bands at approximately 17-30 kDa depending on isoform and processing

  • Flow Cytometry: Identifies IL-37-expressing cells within heterogeneous populations through intracellular staining

  • ELISA: Quantitatively measures IL-37 levels in biological fluids

  • Immunohistochemistry (IHC): Visualizes IL-37 expression patterns in tissue sections

  • Immunocytochemistry (ICC): Detects IL-37 in cultured cells

  • Immunoprecipitation (IP): Isolates IL-37 and associated protein complexes

  • Functional blocking: Neutralizes IL-37 activity to study its biological functions

• What are the key considerations for selecting an appropriate IL-37 antibody?

  When selecting an IL-37 antibody, researchers should consider:

  • Specificity: Whether the antibody recognizes specific isoforms or all IL-37 variants

  • Host species: Compatibility with experimental design (rabbit, mouse, goat, etc.)

  • Clonality: Monoclonal for specific epitopes, polyclonal for broader recognition

  • Validated applications: Confirmed functionality in desired applications (WB, IHC, ELISA, etc.)

  • Conjugation: Whether unconjugated or conjugated to fluorophores/enzymes

  • Cross-reactivity: Specificity for human IL-37 (note: no mouse homolog exists)

  • Recognition region: Whether it targets N-terminal or mature regions (affects detection of processed forms)

  • Validated literature: Previously successful use in published research

Advanced Research Applications and Technical Considerations

• How can IL-37 antibodies be used to investigate both intracellular and extracellular IL-37 functions?

  IL-37 exhibits dual functionality operating through both intracellular and extracellular mechanisms:

  Intracellular function investigation:

  • Immunocytochemistry with nuclear co-localization markers to track nuclear translocation

  • Co-immunoprecipitation with Smad3 to detect IL-37-Smad3 complex formation

  • Subcellular fractionation followed by Western blot to quantify nuclear vs. cytoplasmic IL-37

  • Confocal microscopy with fluorescently labeled antibodies to visualize intracellular trafficking

  Extracellular function investigation:

  • Neutralizing antibodies to block receptor binding and signaling

  • ELISA to measure secreted IL-37 levels in culture supernatants

  • Co-immunoprecipitation with IL-18Rα or IL-1R8 to confirm receptor binding

  • Surface plasmon resonance with purified receptors to measure binding kinetics

  Research has demonstrated that IL-37 functions intracellularly by binding to Smad3 after caspase-1 cleavage and translocating to the nucleus, while extracellularly it forms a complex with IL-18Rα and IL-1R8 to transduce anti-inflammatory signals .

• What methodologies are optimal for detecting IL-37 expression in different cell types and how does activation affect expression?

  Different cell types require optimized detection strategies:

  Cell Type	Optimal Detection Method	Activation Condition	Expression Pattern
  PBMCs	Flow cytometry with intracellular staining	LPS (2 μg/mL, 24h)	Increased from basal levels
  Monocytes	Flow cytometry or PrimeFlow RNA assay	LPS stimulation	~87% express basally, increasing to 97% after LPS
  Treg cells	PrimeFlow RNA assay	LPS stimulation	~80% express basally, increasing to 91% after LPS
  Macrophages (M1)	Western blot/ELISA	LPS challenge	Responsive to IL-37 treatment, reducing inflammation
  Macrophages (M2)	Western blot/ELISA	LPS challenge	Less responsive to IL-37 treatment
  Dendritic cells	Immunocytochemistry	TLR stimulation	Can become tolerogenic upon IL-37 exposure
  THP-1 cells	Western blot	LPS (10 μg/mL, 3h) + PMA (200 nM, 24h)	Inducible expression

  The PrimeFlow RNA assay is particularly valuable for simultaneous measurement of IL-37 mRNA expression in multiple immune cell subsets without culture or fractionation .

• How can researchers develop and validate their own monoclonal antibodies against IL-37?

  Development of monoclonal antibodies against IL-37 involves:

  1. Immunogen preparation:

     • Express soluble human IL-37b protein in E. coli using pET28a/IL-37 expression system

     • Purify the ~25 kDa recombinant protein using appropriate chromatography methods

  2. Immunization:

     • Immunize BALB/c mice with purified recombinant IL-37 protein mixed with Freund's adjuvant

     • Administer booster injections at 2-week intervals

     • Monitor antibody titers by indirect ELISA

  3. Hybridoma generation:

     • Isolate splenocytes from immunized mice

     • Fuse with SP2/0 myeloma cells using 50% polyethylene glycol

     • Culture in HAT medium for selection

  4. Screening:

     • Initial screening by indirect ELISA using prokaryotic expressed soluble IL-37 protein

     • Secondary screening with eukaryotic expressed IL-37-GFP fusion protein

     • Confirm specificity by Western blotting and flow cytometry

  5. Validation:

     • Test antibody against recombinant IL-37 protein

     • Evaluate recognition of native IL-37 in human samples (PBMCs, cell lines)

     • Confirm specificity across multiple applications (WB, ELISA, IHC, flow cytometry)

     • Determine if the antibody recognizes specific isoforms or all IL-37 variants

• What are the complexities in detecting IL-37 expression and activity in human vs. experimental models?

  Key complexities include:

  • Species differences: IL-37 homologous gene has not been identified in mice, requiring generation of transgenic mice expressing human IL-37 (IL-37-tg) for in vivo studies

  • Isoform specificity: The five human IL-37 isoforms (a-e) have different expression patterns and possibly different functions, requiring isoform-specific detection methods

  • Processing dynamics: IL-37 is expressed as an inactive precursor requiring caspase-1 cleavage for activation, creating both full-length and processed forms that may behave differently

  • Concentration-dependent effects: Recombinant IL-37 shows optimal anti-inflammatory activity at low picomolar concentrations rather than nanomolar concentrations, creating a narrow detection window

  • Receptor complexities: IL-37 interacts with multiple receptors (IL-18Rα, IL-1R8) and forms a trimeric complex with IL-18BP and IL-18Rβ, requiring sophisticated approaches to analyze receptor engagement

  • Donor variability: Studies show significant donor variation in IL-37 responsiveness, with only a subgroup of donors (35 out of total tested) showing strong responses to IL-37 treatment

• How can IL-37 antibodies be used to investigate the mechanisms of IL-37-mediated immune regulation?

  IL-37 antibodies enable investigation of multiple regulatory mechanisms:

  • Cytokine suppression pathways: Neutralizing IL-37 antibodies can reverse the inhibition of pro-inflammatory cytokines (IL-1β, IL-6, TNFα), revealing the dependency of inflammatory suppression on IL-37

  • Signaling pathway analysis: Using IL-37 antibodies in phospho-flow or Western blot experiments to examine how IL-37 reduces LPS-induced p38 and pERK activation

  • Immune cell polarization: Tracking how IL-37 impacts macrophage polarization between M1/M2 phenotypes using flow cytometry and cytokine profiling

  • T-cell differentiation: Using IL-37 antibodies to understand how IL-37 regulates Th1, Th2, and Th17 responses and promotes regulatory T-cell development

  • Receptor engagement: Co-immunoprecipitation with IL-37 antibodies to identify IL-37's interaction with IL-18Rα and IL-1R8 receptors

  • Smad3 dependency: IL-37 antibodies can help investigate how IL-37 interacts with Smad3 and promotes nuclear translocation of pSmad3, as demonstrated with IL-37d

• What experimental approaches can determine if IL-37 functions differently across various disease models?

  To investigate differential functions of IL-37 across disease models:

  • Comparative disease models: Use IL-37 antibodies to quantify expression and localization across multiple disease models (endotoxemia, asthma, inflammatory bowel disease, cancer)

  • Tissue-specific analysis: Perform immunohistochemistry with IL-37 antibodies on tissues from different disease contexts to identify tissue-specific expression patterns

  • Cytokine profiling: Use antibody arrays to identify disease-specific changes in cytokine profiles after IL-37 administration, as demonstrated in asthma models showing reductions in CCL3, CCL4, CCL5

  • Transgenic models: Compare phenotypes of IL-37 transgenic mice challenged with different disease stimuli (LPS, allergens, autoimmune triggers)

  • Receptor dependency: Use IL-1R8-deficient models to assess whether IL-37's function depends on the same receptors across different disease states

  • Isoform specificity: Test if different IL-37 isoforms (a-e) have differential effects in various disease models using isoform-specific antibodies

  For example, in asthma models, IL-37 reduces symptoms by inhibiting pro-inflammatory cytokines like CCL3, CCL4, and CCL5 , while in endotoxemia models, IL-37 suppresses multiple inflammatory pathways including TNF signaling, NOD-like receptor signaling, and NF-kappa B signaling .

• How can researchers ensure the specificity and validity of their IL-37 antibody-based experiments?

  Critical validation steps include:

  • Positive controls: Use recombinant IL-37 protein or LPS-stimulated PBMCs/THP-1 cells known to express IL-37

  • Negative controls: Include IL-1R8-deficient cells, which should not respond to IL-37 treatment

  • Isotype controls: Use matched isotype IgG controls to ensure specific immunostaining in flow cytometry and IHC/ICC applications

  • Peptide competition: Pre-incubate antibody with recombinant IL-37 peptide to confirm binding specificity

  • Multiple antibody validation: Use two different antibodies targeting different epitopes of IL-37

  • Knockout/knockdown validation: Use siRNA knockdown of IL-37 or cells from IL-37 transgenic models as specificity controls

  • Cross-reactivity testing: Confirm the antibody does not cross-react with other IL-1 family members by testing against recombinant proteins

  • Concentration optimization: Titrate antibody concentrations, particularly for IL-37 neutralizing experiments, as IL-37 shows optimal activity at low picomolar concentrations