IL17RA Antibody

What is IL17RA and why is it significant in immunological research?

IL17RA (Interleukin-17 Receptor A) is a ubiquitous type I membrane glycoprotein that functions as a critical receptor for IL-17A and IL-17F cytokines. It plays essential roles in both innate and adaptive immunity, particularly in responses against fungal pathogens such as Candida albicans. IL17RA forms part of a receptor complex that mediates downstream signaling upon binding with IL-17 family cytokines, activating inflammatory pathways and promoting neutrophil recruitment and maturation.

The receptor is particularly significant because it represents a crucial mediator in the pathogenesis of several inflammatory and autoimmune diseases, including psoriasis and rheumatoid arthritis. Additionally, defects in IL17RA have been identified as the cause of familial candidiasis type 5 (CANDF5), a rare disorder characterized by altered immune responses and impaired clearance of fungal infections . Understanding IL17RA function through antibody-based approaches has therefore become central to both basic immunology research and translational medicine.

What experimental approaches utilize IL17RA antibodies in research settings?

IL17RA antibodies are employed in numerous experimental techniques, each providing distinct insights into receptor biology and function:

• Flow Cytometry: IL17RA antibodies are used to quantitatively determine receptor density on cell surfaces. For example, researchers have employed anti-IL17RA antibodies conjugated with fluorescent markers like carboxyfluorescein to analyze receptor expression on various cell populations, including monocytes and T cells .

• Immunohistochemistry/Immunofluorescence: These techniques allow visualization of IL17RA distribution in tissues and cells. Researchers frequently use primary antibodies against IL17RA followed by fluorophore-conjugated secondary antibodies to detect expression patterns in specific cell types, as demonstrated in studies examining IL17RA in CNS tissues during EAE .

• Western Blotting: IL17RA antibodies enable detection of receptor protein expression in cell and tissue lysates, providing information about protein size and potential post-translational modifications.

• Immunoprecipitation: Anti-IL17RA antibodies can precipitate the receptor and its binding partners from cell lysates, allowing researchers to study protein-protein interactions. This approach has been used to investigate interactions between IL17RA and other components of the signaling complex, such as IL17RC and IL17RD .

• Blocking Experiments: Neutralizing IL17RA antibodies are valuable for studying the functional consequences of disrupting IL-17 signaling pathways in both in vitro and in vivo systems .

How should researchers validate IL17RA antibodies for their specific applications?

Proper validation of IL17RA antibodies is critical for ensuring experimental reliability and reproducibility. Researchers should implement the following validation strategies:

• Cross-reactivity Testing: Verify antibody specificity by confirming absence of cross-reactivity with related proteins. For instance, high-quality IL17RA antibodies should not cross-react with other IL-17 receptor family members such as IL-17B receptor, as demonstrated in direct ELISA assays .

• Positive and Negative Controls: Include appropriate controls in all experiments. Positive controls might include cell lines known to express high levels of IL17RA, while negative controls could utilize cells where IL17RA expression is knocked down or naturally absent.

• Multiple Detection Methods: Validate findings using complementary techniques. For example, combining flow cytometry with immunohistochemistry or Western blotting provides more robust evidence of antibody specificity.

• Genetic Controls: When possible, utilize IL17RA knockout cells or tissues as negative controls to conclusively demonstrate antibody specificity.

• Isotype Controls: Always include appropriate isotype-matched control antibodies to distinguish specific binding from non-specific background, as demonstrated in flow cytometry protocols where protein G-purified normal goat-IgG conjugated with carboxyfluorescein was used as an isotype control for IL17RA staining .

• Antibody Titration: Determine optimal antibody concentration through titration experiments to maximize signal-to-noise ratio for each specific application.

What are the critical technical considerations for IL17RA antibody preparation and storage?

Proper handling of IL17RA antibodies significantly impacts experimental outcomes. Researchers should consider the following technical aspects:

• Reconstitution Protocol: Follow manufacturer-specific guidelines for reconstitution. For instance, some IL17RA antibodies require reconstitution at 0.5 mg/mL in sterile PBS to maintain optimal activity .

• Endotoxin Levels: For functional studies and in vivo applications, verify that antibody preparations have acceptably low endotoxin levels (e.g., <0.10 EU per 1 μg of antibody) .

• Storage Conditions: Store reconstituted antibodies according to manufacturer recommendations, typically at 2-8°C for short-term storage or aliquoted and stored at -20°C or -80°C for long-term preservation.

• Freeze-Thaw Cycles: Minimize freeze-thaw cycles as repeated freezing and thawing can compromise antibody function and specificity.

• Buffer Compatibility: Ensure that the buffer used for antibody dilution is compatible with the intended application. For example, certain buffers containing sodium azide may interfere with some enzymatic assays or in vivo experiments.

• Working Dilution Preparation: Prepare working dilutions immediately before use and maintain appropriate temperature conditions during experimental procedures.

How can IL17RA antibodies be used to investigate IL-17 signaling in fungal immunity models?

IL17RA antibodies serve as powerful tools for dissecting the complex roles of IL-17 signaling in antifungal immunity, particularly against Candida albicans. Researchers can implement several sophisticated approaches:

• Targeted Signaling Pathway Blockade: Neutralizing IL17RA antibodies can be administered in experimental models to specifically block receptor function. For instance, researchers have injected mice intraperitoneally with anti-IL17RA antibodies (clone 657603) at doses of 100–500 μg/injection on days -1, +1, and +2 relative to Candida infection to evaluate the receptor's role in oral candidiasis .

• Differential Blockade Strategy: Comparing the effects of blocking different components of the IL-17 pathway can reveal functional redundancy. Studies have shown that while IL-17A blockade predisposes mice to oropharyngeal candidiasis (OPC), IL-17F blockade does not have the same effect, highlighting the specific role of IL-17A-IL17RA interaction in mucosal fungal defense .

• Ex Vivo Restimulation Assays: IL17RA antibodies can be used to assess recall responses in cells isolated from infected animals. Researchers have cultured cervical lymph node cells with heat-killed C. albicans in the presence or absence of IL17RA blocking antibodies to evaluate cytokine production patterns and memory responses .

• Combinatorial Cytokine Blockade: Advanced studies can combine IL17RA antibodies with blockers of other inflammatory cytokines to identify synergistic or compensatory immune mechanisms in fungal defense.

• Tissue-Specific Deletion Models: Complementing antibody blockade studies with tissue-specific genetic deletion models of IL17RA provides comprehensive insights into compartmentalized immune responses against fungi.

What methodological differences should researchers consider when using IL17RA antibodies for flow cytometry versus immunohistochemistry?

Different experimental applications require specific methodological considerations for optimal results:

For Flow Cytometry:

• Antibody Conjugation: Use directly conjugated antibodies (e.g., APC, PE, or FITC-labeled) or implement a two-step staining approach with appropriate fluorochrome-conjugated secondary antibodies for maximum sensitivity.

• Cell Preparation Protocol: For optimal detection of IL17RA, researchers should:

  • Perform FC-blocking (e.g., with 1 μg of mouse IgG per 10^6 cells for 15 minutes) before staining to minimize non-specific binding .

  • Consider using unfixed cells for surface staining as fixation may alter epitope accessibility.

  • When analyzing specific cell populations, implement multi-color panels with lineage markers (e.g., CD3 for T cells, CD14 for monocytes) to accurately identify IL17RA expression on different cell types .

• Controls and Gating Strategy: Include fluorescence-minus-one (FMO) controls and implement consistent gating strategies based on forward/side scatter profiles and lineage markers before analyzing IL17RA expression.

For Immunohistochemistry/Immunofluorescence:

• Fixation Protocol: Optimize fixation conditions as excessive fixation may mask IL17RA epitopes. Studies have employed 2% paraformaldehyde for 10 minutes when working with cultured cells .

• Antigen Retrieval: For tissue sections, appropriate antigen retrieval methods may be necessary to expose IL17RA epitopes that could be masked during fixation.

• Double/Triple Labeling: For co-localization studies, carefully select compatible antibody combinations. Researchers have successfully performed double immunofluorescence using anti-GFAP monoclonal antibodies in combination with anti-IL17R antibodies to identify IL17RA expression in astrocytes .

• Detection Systems: Choose appropriate detection systems based on desired sensitivity. Amplification methods like tyramide signal amplification may be beneficial for detecting low-abundance IL17RA expression.

• Background Reduction: Implement appropriate blocking steps (e.g., with serum from the same species as the secondary antibody) to minimize non-specific background staining.

How do mutations in IL17RA affect antibody binding and experimental interpretations?

Mutations in IL17RA present significant challenges for antibody-based research, requiring careful experimental design and interpretation:

What are the latest applications of IL17RA antibodies in studying autoimmune conditions?

IL17RA antibodies have become instrumental in investigating the complex pathophysiology of autoimmune disorders:

• CNS Inflammation Models: In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, IL17RA antibodies have been used to demonstrate increased receptor expression in the central nervous system compared to healthy controls. This approach has revealed that IL17RA signaling in glial cells may play an important role in neuroinflammatory processes .

• Psoriasis Research: Recent studies have employed IL17RA antibodies to investigate the dual pathophysiology of psoriasis and chronic mucocutaneous candidiasis. Transcriptomic analysis of skin biopsies from patients with IL17RA mutations revealed distinct psoriasis-associated signatures intertwined with inflammatory pathways related to fungal infections, providing insights into shared molecular mechanisms .

• Neutrophil Function Analysis: Given that IL-17A is a potent inducer of the maturation of CD34-positive hematopoietic precursors into neutrophils, IL17RA antibodies are being used to investigate how receptor signaling influences neutrophil development and function in autoimmune conditions .

• Germinal Center Reactions: IL17RA antibodies have been employed to explore the receptor's essential role in the optimal localization of follicular T helper cells within lymphoid tissues, revealing how IL-17 serves as an extrinsic stop signal that acts on post-differentiated IL-17RA+ T follicular helper cells to enable their interaction with responder B cells in the light zone niche .

• Therapy Response Prediction: Researchers are developing applications of IL17RA antibodies to predict patient responses to IL-17 pathway-targeting biologics, which are increasingly used for treating conditions like psoriasis, psoriatic arthritis, and ankylosing spondylitis .

How can researchers address potential cross-reactivity concerns with IL17RA antibodies?

Cross-reactivity represents a significant challenge in antibody-based research. To mitigate this issue with IL17RA antibodies, researchers should implement the following strategies:

• Comprehensive Cross-Reactivity Testing: Validate antibodies against related receptor family members. High-quality IL17RA antibodies should demonstrate no cross-reactivity with recombinant mouse IL-17R, mouse IL-17BR, or human IL-17BR in direct ELISAs and Western blots .

• Multi-technique Validation: Confirm antibody specificity using complementary techniques. For example, if cross-reactivity is absent in ELISA but present in Western blot, this suggests conformation-dependent specificity that researchers must account for in experimental design.

• Genetic Controls: When available, utilize IL17RA knockout or knockdown systems as definitive negative controls. Studies have employed IL-17A^(-/-), IL-17F^(-/-), and Act1^(-/-) mice with age- and sex-matched controls to validate antibody specificity in vivo .

• Species-Specific Validation: When working across species, validate antibody specificity for each target organism. Some antibodies may recognize human IL17RA but fail to detect the murine ortholog due to structural differences.

• Antibody Fragment Utilization: In certain applications, using F(ab) or F(ab')2 fragments instead of whole IgG molecules can reduce non-specific binding mediated by Fc receptors.

• Competitive Binding Assays: Perform pre-absorption with recombinant IL17RA protein before staining to demonstrate binding specificity through signal reduction or elimination.