IL17A Recombinant Monoclonal Antibody

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

IL-17A (also known as CTLA-8) is a 32 kDa disulfide-linked homodimer consisting of 136 amino acids that functions as a CD4+ T cell-derived cytokine. It promotes inflammatory responses and is elevated in numerous pathological conditions including rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, and transplant rejection. IL-17A belongs to a family of six related proteins (IL-17A through IL-17F) and signals through the IL-17 receptor (IL-17R/CDw217). Its central role in mediating inflammatory responses makes it a crucial target for immunological research, particularly for developing therapeutic approaches for autoimmune and inflammatory disorders .

How do recombinant monoclonal antibodies differ from traditional monoclonal antibodies?

Recombinant monoclonal antibodies are produced using in vitro expression systems rather than hybridoma technology. These antibodies are generated by cloning specific genes that code for the desired antibody into an expression vector, which is then expressed in vitro. The key advantages include significantly better specificity and superior lot-to-lot consistency compared to traditional monoclonal antibodies. This manufacturing process allows for precise genetic manipulation to enhance binding affinity, species cross-reactivity, or introduce specific conjugation sites. Additionally, the recombinant approach eliminates concerns related to hybridoma instability or animal welfare considerations associated with traditional methods .

What are the primary research applications for IL17A recombinant monoclonal antibodies?

IL17A recombinant monoclonal antibodies are utilized across multiple research applications including:

• Western Blot (WB) for protein detection and quantification

• Immunohistochemistry (IHC) for tissue localization

• Flow Cytometry for cellular analysis, particularly intracellular staining

• Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection

• Immunofluorescence (IF) for subcellular localization

Each application requires specific optimization protocols, with particular attention to antibody concentration and buffer conditions. For intracellular staining applications, appropriate fixation and permeabilization methods are crucial for accessing the target protein while maintaining cellular architecture .

How should researchers address species cross-reactivity when using IL17A antibodies across different experimental models?

When working with IL17A antibodies across different species models, researchers must carefully validate cross-reactivity profiles. Based on available data, human IL17A antibodies may show variable cross-reactivity with mouse and rat orthologs. For instance, certain clones show 100% cross-reactivity with recombinant human (rh) IL-17A/IL-17F heterodimer but no cross-reactivity with rhIL-17F, recombinant mouse IL-17, or recombinant rat IL-17A .

To address this challenge:

• Perform initial validation experiments with positive controls from each species

• Consider using species-specific antibodies when possible

• For cross-species studies, select antibodies raised against conserved epitopes

• Document exact clone numbers and validation data in your research protocols

• Utilize Western blotting with recombinant proteins to confirm specificity before proceeding to more complex applications

This approach prevents misinterpretation of data due to unexpected cross-reactivity patterns or lack of reactivity in certain species .

What are the optimal experimental conditions for detecting low levels of IL17A expression in immune cells?

Detecting low levels of IL17A expression in immune cells requires careful optimization of experimental conditions:

• Cell Stimulation Protocol: Treat peripheral blood mononuclear cells (PBMCs) with 50 ng/mL PMA and 250 ng/mL calcium ionomycin for 16 hours to upregulate IL-17A expression before antibody staining .

• Fixation and Permeabilization: Use specialized fixation buffers followed by permeabilization solutions designed for intracellular cytokine detection. Standard protocols often fail to adequately expose intracellular epitopes of IL-17A.

• Antibody Titration: Determine the optimal antibody concentration through titration experiments (typically starting at 1 μg/mL for Western blot applications) .

• Signal Amplification: For very low expression levels, consider employing signal amplification systems such as tyramide signal amplification or polymer-based detection systems.

• Controls: Always include appropriate positive controls (stimulated T cells) and negative controls (isotype antibodies matched to your primary antibody) .

These methodological refinements can significantly improve the signal-to-noise ratio when detecting limited IL17A expression in primary immune cells or tissue samples.

How can researchers differentiate between detection of IL17A homodimers versus IL17A/F heterodimers in experimental systems?

Differentiating between IL17A homodimers and IL17A/F heterodimers requires strategic experimental design:

• Antibody Selection: Choose antibodies with documented specificity profiles. For example, certain IL17A antibodies show 100% cross-reactivity with IL-17A/IL-17F heterodimers while showing no cross-reactivity with IL-17F homodimers .

• Sequential Immunoprecipitation: Perform initial immunoprecipitation with anti-IL17A, followed by Western blotting with anti-IL17F (and vice versa) to identify heterodimers.

• Molecular Weight Analysis: Under reducing conditions, IL17A appears at approximately 17 kDa on Western blots, while IL17A/F heterodimers may show distinct banding patterns between 14-19 kDa .

• Recombinant Protein Controls: Include purified recombinant IL17A, IL17F, and IL17A/F heterodimers as controls in your experiments to establish distinct detection patterns.

• Mass Spectrometry: For definitive differentiation, consider targeted mass spectrometry approaches after immunoprecipitation to identify specific peptide sequences unique to each dimer configuration.

This multi-faceted approach enables researchers to accurately distinguish between these structurally similar but functionally distinct cytokine configurations .

What optimization strategies should be employed when using IL17A antibodies for Western blot analysis?

Successful Western blot analysis with IL17A antibodies requires several critical optimization steps:

• Sample Preparation: For primary human samples, CD4+ T cells must be properly activated to express detectable IL17A. For recombinant systems, transfection efficiency should be verified.

• Reduction Conditions: Use reducing conditions (typically including β-mercaptoethanol or DTT) as IL17A contains disulfide bonds that affect epitope accessibility. Documented protocols confirm detection under reducing conditions using Immunoblot Buffer Group 1 .

• Antibody Concentration: Start with 1 μg/mL of anti-IL17A monoclonal antibody, followed by appropriate HRP-conjugated secondary antibody .

• Membrane Selection: PVDF membranes have demonstrated superior results compared to nitrocellulose for IL17A detection .

• Expected Molecular Weight: Look for specific bands at approximately 17 kDa for native IL17A, though in transfection systems bands may appear between 14-19 kDa due to variations in post-translational modifications .

• Controls: Include positive controls (stimulated CD4+ T cells) and negative controls (mock transfected cells) to verify specificity .

These methodological refinements significantly improve reproducibility and sensitivity when detecting IL17A via Western blot.

What are the critical parameters for successful intracellular staining of IL17A in flow cytometry experiments?

Intracellular staining for IL17A requires attention to several critical parameters:

• Cell Stimulation: Stimulate PBMCs with 50 ng/mL PMA and 250 ng/mL calcium ionomycin for 16 hours to upregulate IL17A expression .

• Fixation Protocol: Use specialized flow cytometry fixation buffers that maintain cellular architecture while enabling antibody access to intracellular targets .

• Permeabilization Strategy: Apply permeabilization/wash buffers specifically designed for intracellular cytokine detection rather than general permeabilization reagents .

• Antibody Selection: Choose antibodies validated specifically for flow cytometry applications, as not all IL17A antibodies perform equally across different applications .

• Controls: Include unstimulated cells as negative controls, isotype controls to assess non-specific binding, and fluorescence-minus-one (FMO) controls for accurate gating strategies .

• Multicolor Panel Design: When incorporating IL17A detection into multicolor panels, consider spectral overlap and compensation requirements, particularly when using bright fluorophores.

Adherence to these methodological guidelines enables reliable detection of IL17A-producing cells in complex samples like PBMCs or tissue digests.

How should researchers validate the specificity of IL17A recombinant monoclonal antibodies before experimental use?

Comprehensive validation of IL17A recombinant monoclonal antibodies should follow this multi-step approach:

• ELISA Validation: Perform direct ELISAs against recombinant IL17A, IL17F, and IL17A/F heterodimers to establish specificity profiles. Document cross-reactivity percentages with related proteins .

• Western Blot Verification: Test antibodies against both recombinant proteins and biological samples (e.g., activated T cells known to express IL17A) .

• Knockout/Knockdown Controls: When possible, include IL17A knockout cells or knockdown systems as negative controls to confirm specificity.

• Epitope Mapping: Determine the specific epitope recognized by the antibody to predict potential cross-reactivity with related proteins.

• Blocking Experiments: Perform pre-adsorption experiments with recombinant IL17A to demonstrate that binding can be specifically blocked.

• Species Cross-Reactivity Assessment: Systematically test against IL17A from different species if cross-species applications are intended .

This comprehensive validation strategy ensures experimental reliability and facilitates accurate interpretation of results when using these antibodies in research applications.

What is the molecular structure and functional domain organization of IL17A relevant to antibody targeting?

IL17A has a distinct molecular structure that influences antibody targeting strategies:

Understanding these structural features is essential for selecting appropriate antibodies and interpreting experimental results. Most validated IL17A recombinant monoclonal antibodies target epitopes within the IL17 domain (Ile20-Ala155), which contains the functionally important receptor-binding regions .

What are the comparative characteristics of different IL17A recombinant monoclonal antibody clones available for research?

Different IL17A recombinant monoclonal antibody clones exhibit distinct characteristics important for experimental design:

This comparative analysis highlights the importance of selecting the appropriate clone based on your specific experimental requirements, including species of interest, application, and need for distinguishing between IL17A homodimers and IL17A/F heterodimers .

What signal transduction pathways are regulated by IL17A that can be monitored as functional readouts of antibody efficacy?

IL17A regulates multiple signal transduction pathways that can serve as functional readouts for antibody efficacy:

These pathways provide functional validation of antibody efficacy beyond simple binding assays. When testing neutralizing antibodies against IL17A, researchers should monitor at least two independent signaling outputs to confirm complete blockade of biological activity .

What are the key considerations for researchers selecting IL17A recombinant monoclonal antibodies for their studies?

Researchers selecting IL17A recombinant monoclonal antibodies should consider several critical factors to ensure experimental success:

• Application Suitability: Different antibody clones perform optimally in specific applications. Verify that the antibody has been validated for your intended use (Western blot, flow cytometry, IHC, etc.) .

• Species Reactivity: Confirm that the antibody recognizes IL17A from your species of interest. Many antibodies exhibit restricted species reactivity profiles .

• Specificity Profile: Determine whether the antibody can distinguish between IL17A homodimers and IL17A/F heterodimers if this distinction is relevant to your research question .

• Clone Characteristics: Recombinant monoclonal antibodies offer superior lot-to-lot consistency and specificity compared to other antibody types, making them preferable for longitudinal studies or quantitative applications .

• Validation Data: Review available validation data, including Western blot images, ELISA cross-reactivity profiles, and flow cytometry histograms to ensure the antibody performs as expected .

• Titration Requirements: All antibodies require careful titration for optimal performance. Budget time for this optimization step in your experimental planning .