IL17A Antibody

What is IL-17A and why is it a target for antibody research?

IL-17A is a CD4+ T cell-derived proinflammatory cytokine that plays a critical role in immune regulation. It exists primarily as glycosylated 20-30 kD homodimers and promotes inflammatory responses in various cell lines. IL-17A is elevated in multiple inflammatory conditions including rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, and transplant rejection, making it an important therapeutic target . IL-17A signals through the IL-17RA-IL-17RC heterodimeric receptor complex, triggering homotypic interaction with TRAF3IP2 adapter, leading to downstream TRAF6-mediated activation of NF-kappa-B and MAP kinase pathways .

How do IL-17A antibodies differ in their specificity for IL-17 family members?

IL-17A antibodies vary significantly in their cross-reactivity with other IL-17 family members. For example, the eBio17B7 antibody reacts with mouse and rat IL-17A but shows no recognition of IL-17F . Some antibodies like 496.g1 demonstrate binding to both IL-17A (EC₉₀ 12.1 ng/mL) and IL-17F (EC₉₀ 358.5 ng/mL) with little or no binding to human IL-17B, IL-17C, IL-17D, and IL-17E . More specialized dual-targeting antibodies, such as 496.g3 (bimekizumab), have been engineered to bind both IL-17A and IL-17F with high affinity (KD values of 7 pM and 35 pM, respectively) .

What applications are commonly used for IL-17A antibodies in research?

IL-17A antibodies have been validated for multiple research applications:

Sources:

How can I optimize fixation and permeabilization for intracellular IL-17A staining?

For optimal intracellular staining of IL-17A, cells should be stimulated with PMA (50 ng/mL) and calcium ionomycin (250 ng/mL) for 16-18 hours to induce cytokine production. After stimulation, cells should be fixed with paraformaldehyde and permeabilized with saponin or a dedicated permeabilization buffer like Flow Cytometry Permeabilization/Wash Buffer I . For tissue samples, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 can be used alternatively . The staining procedure should include appropriate blocking steps to reduce non-specific binding and use recommended antibody dilutions (typically 1:200-1:800 for immunofluorescence) .

How do IL-17A neutralizing antibodies differentially affect IL-17A, IL-17F, and IL-17AF heterodimers in functional assays?

Research demonstrates distinct neutralization profiles for different anti-IL-17A antibodies. Anti-IL-17A antibodies that neutralize both IL-17A and IL-17AF caused elevated oral fungal loads in mouse models of oropharyngeal candidiasis, whereas anti-IL-17AF and anti-IL-17F antibodies alone did not . This suggests a hierarchy of functional importance among these cytokine variants.

In cellular assays, anti-IL-17A antibodies can block signaling induced by IL-17A and IL-17AF but not IL-17F, while anti-IL-17F antibodies efficiently block signaling by IL-17F but not IL-17A or IL-17AF . For example, in normal human dermal fibroblasts (NHDFs) stimulated with TNFα and IL-17A or IL-17F, the neutralization potency varies significantly between antibodies. The dual-targeting antibody 496.g3 demonstrated improved neutralization of IL-17F-induced IL-6 production compared to the IL-17A-preferential antibody 496.g1 .

What experimental approaches can resolve contradictory findings when using IL-17A antibodies versus genetic knockout models?

Researchers often encounter discrepancies between antibody neutralization and genetic knockout results. To resolve such contradictions, several approaches are recommended:

• Temporal considerations: Unlike genetic knockouts with permanent cytokine deficiency, antibodies provide temporal inhibition. Experiments showed that termination of anti-IL-17A treatment was associated with rapid clearance of Candida albicans infection .

• Combination approaches: Use both approaches in parallel studies. For example, comparing anti-IL-17A antibody treatment with IL-17A-/-, IL-17F-/-, and Act1-/- mice has revealed cooperative effects among IL-17 family cytokines .

• Heterodimer analysis: Since IL-17A can form heterodimers with IL-17F, researchers should evaluate the specificity of their antibodies against homodimers versus heterodimers using techniques like surface plasmon resonance (SPR) .

• Comprehensive cytokine profiling: Measure multiple downstream cytokines (IL-6, IL-8, G-CSF, PGE2) to gain a complete picture of pathway inhibition .

How can I engineer improved IL-17A antibodies with enhanced affinity and specificity?

Engineering enhanced IL-17A antibodies involves several sophisticated approaches:

• Affinity maturation: As demonstrated with the development of 496.g3 from 496.g1, introducing five specific mutations in the light chain variable region increased binding affinity for IL-17F by 43-fold (from KD 1510 pM to 35 pM) and for IL-17A by 4-fold (from KD 29 pM to 7 pM) .

• In silico design methods: Applied to the antibody-antigen interface, computational approaches can identify mutation combinations that simultaneously improve binding to multiple targets .

• Single B cell selection methods: This approach has been successful in identifying parental antibodies with strong binding properties that can be further optimized .

• Stable cell line development: For consistent antibody production, generating stable HEK293-F cell lines expressing the antibody fragment through G418 selection of transfected cells has proven effective .

What methodological considerations are critical when testing IL-17A antibodies in animal models of inflammatory disease?

When designing animal studies with IL-17A antibodies, several critical factors must be considered:

• Species cross-reactivity: Confirm the antibody's reactivity with the experimental species. For example, HAP peptide has >10-fold weaker affinity for mouse IL-17A compared to human IL-17A .

• Dosing regimen: Successful protocols typically involve multiple antibody injections. For oropharyngeal candidiasis studies, mice received 100–500 μg/injection intraperitoneally on days -1, +1, and +2 relative to infection .

• Relative cytokine potency: Consider species differences in cytokine potency. The relative difference in potency between human IL-17A and IL-17F is ~100-fold, whereas in mice it's 10,000-fold .

• Readout selection: Choose appropriate inflammatory markers. For IL-17A neutralization in mouse models, measuring plasma KC (keratinocyte) levels provides a sensitive readout of antibody efficacy .

• Disease model selection: For psoriasis research, the imiquimod-induced psoriasis mouse model has been validated for evaluating IL-17A antibody efficacy through measurement of the psoriasis index .

How can I improve reproducibility when refolding recombinant IL-17A for antibody binding studies?

Refolding recombinant IL-17A can be challenging. An optimized protocol involves:

• Two-way optimization: Systematically explore various ratios of oxidized and reduced forms of oxido-shuffling reagents and refolding duration .

• Expression system selection: Escherichia coli systems have been successfully used for IL-17A expression, though careful attention to inclusion body processing is required .

• Standardized protocol adoption: Follow validated protocols such as those submitted to protocols.io (https://www.protocols.io/view/optimized-refolding-protocol-for-il-17a-bda3i2gn)[4].

• Complex formation: For difficult applications like crystallization, forming complexes with Fab fragments or high-affinity peptides can stabilize IL-17A structure .

What controls should be implemented when validating IL-17A antibody specificity in flow cytometry?

Rigorous validation of IL-17A antibodies for flow cytometry requires multiple controls:

• Stimulation controls: Compare unstimulated cells with those stimulated with PMA (50 ng/mL) and calcium ionomycin (250 ng/mL) for 16 hours .

• Isotype controls: Include proper isotype-matched control antibodies (e.g., IgG2a clone 54447, IgG1 clone 43414) at the same concentration as the test antibody .

• Blocking experiments: Pre-incubate the antibody with recombinant IL-17A before staining to confirm binding specificity .

• Cross-reactivity assessment: Test against cells expressing related cytokines to ensure specificity within the IL-17 family .

• Knockout/knockdown validation: When possible, include IL-17A knockout/knockdown samples as negative controls .

How can I optimize IL-17A detection in different tissue types for immunohistochemistry?

Optimizing IL-17A detection in tissues requires specific considerations:

• Tissue-specific protocols: Different tissues require adjusted protocols. For example:

  • Human tonsil: 1 μg/mL antibody concentration, DAB (brown) staining, hematoxylin (blue) counterstain

  • Crohn's disease intestine: 0.5-25 μg/mL antibody concentration

  • Skin tissues: TE buffer pH 9.0 for antigen retrieval

• Cell-type identification: Combine IL-17A staining with lineage markers. For instance, in acne vulgaris lesions, double staining for IL-17A (green) and CD3 (red) identifies IL-17A-producing T cells .

• Signal amplification: For low-expression tissues, consider using polymer-based detection systems like Anti-Goat IgG VisUCyte™ HRP Polymer .

• Positive tissue selection: Include known positive tissues as controls; human tonsillitis tissue and psoriatic skin lesions consistently show IL-17A expression .

What factors influence the variation in molecular weight detection of IL-17A in Western blot analysis?

Researchers often observe IL-17A at different molecular weights in Western blot analyses due to several factors:

• Glycosylation status: IL-17A exists as glycosylated 20-30 kD homodimers, and the degree of glycosylation can vary between cell types and expression systems .

• Dimerization state: Under reducing conditions, IL-17A typically appears at approximately 15-19 kDa (monomer), while non-reducing conditions may show bands at ~30-40 kDa (dimer) .

• Protein source: Recombinant protein may differ from naturally produced IL-17A. For example, E. coli-derived recombinant human IL-17A (Ile20-Ala155) will have a different molecular weight than full-length glycosylated protein from human cells .

• Sample preparation: Different lysis and sample preparation methods can affect observed molecular weights. Compare whole cell lysates with conditioned-media supernatants to identify secreted versus intracellular forms .

• Antibody clone specificity: Different antibody clones may recognize distinct epitopes that are differentially exposed under various conditions, affecting apparent molecular weight .

How might next-generation IL-17A antibodies address current limitations in therapeutic applications?

Next-generation IL-17A antibodies are being developed to address several limitations:

• Dual-targeting approaches: Following the success of bimekizumab, which targets both IL-17A and IL-17F, future antibodies may target multiple inflammatory mediators simultaneously .

• Enhanced tissue penetration: Developing antibody fragments or alternative scaffolds with improved tissue distribution could enhance efficacy in conditions like psoriatic plaques .

• Reduced immunogenicity: Further humanization and deimmunization strategies might reduce anti-drug antibody responses in patients receiving long-term therapy .

• Subpopulation targeting: Developing antibodies that selectively target IL-17A produced by specific cell populations (Th17 cells versus innate immune sources) could provide more selective immunomodulation .

• Biomarker integration: Developing companion diagnostics to identify patients most likely to respond to IL-17A antibody therapy based on cytokine profiles or genetic markers .

What methodological advances could improve detection sensitivity for low-abundance IL-17A in biological samples?

Several emerging methodological approaches could enhance IL-17A detection sensitivity:

• Single-cell cytokine secretion assays: For detecting IL-17A production at the single-cell level in heterogeneous populations .

• Digital ELISA platforms: Ultra-sensitive detection methods with femtomolar sensitivity for measuring circulating IL-17A in serum/plasma samples .

• Proximity ligation assays: For detecting IL-17A in tissue sections with enhanced spatial resolution and sensitivity .

• Mass cytometry (CyTOF): For simultaneous detection of IL-17A with dozens of other proteins at single-cell resolution .

• Engineered reporter cell lines: Development of sensitive bioassays using cells engineered to produce luciferase or fluorescent proteins in response to IL-17A receptor activation .