IL1RN Antibody

What is IL1RN and what is its biological significance?

Interleukin 1 Receptor Antagonist (IL1RN) is a member of the IL-1 family, which includes the structurally related proteins IL-1α and IL-1β. Unlike other family members, IL1RN functions as a competitive inhibitor of IL-1α and IL-1β by binding to the same receptor without inducing signaling . This antagonistic activity is critical for maintaining homeostasis in inflammatory responses. IL1RN has numerous synonyms in the literature including IL1RA, IL-1ra, IL-1RN, ICIL-1RA, and IRAP (Interleukin 1 receptor antagonist protein) . The biological significance of IL1RN lies in its ability to modulate inflammatory processes by preventing excessive IL-1 signaling, which would otherwise lead to pathological inflammation. Various studies have demonstrated that IL1RN expression levels correlate with inflammatory disease progression, particularly in conditions like interstitial lung disease (ILD) . The balance between IL-1 and IL1RN activity appears to be critical, as patients with specific IL1RN polymorphisms show a higher risk for developing fibrosing alveolitis .

What types of IL1RN antibodies are available for research applications?

Several types of IL1RN antibodies are available for research applications, varying in host species, clonality, and target epitopes. Polyclonal antibodies raised in rabbits are commonly used, such as the ABIN5706884 antibody which recognizes human IL1RN . Both monoclonal and polyclonal antibodies targeting different epitopes of IL1RN exist, including those specific to the C-terminal region (AA 147-177) and those recognizing a broader region (AA 26-177) . Additionally, researchers can choose between unconjugated antibodies and those conjugated to reporter molecules depending on their experimental needs. Host species include rabbit (most common for polyclonals) and mouse (for some monoclonals) . The choice between these different antibody types depends on the specific research application, with polyclonal antibodies often preferred for detection purposes due to their recognition of multiple epitopes, while monoclonals may offer greater specificity for certain applications.

What are the common applications of IL1RN antibodies in research?

IL1RN antibodies are utilized across multiple research applications focusing on inflammatory processes and cytokine biology. Western blotting (WB) represents one of the most common applications, allowing researchers to detect and quantify IL1RN protein expression in cell and tissue lysates . Enzyme-linked immunosorbent assays (ELISA) provide a sensitive method for measuring IL1RN concentrations in biological fluids and conditioned media . Immunocytochemistry (ICC) and immunofluorescence (IF) enable visualization of IL1RN's subcellular localization and expression patterns in cultured cells . Immunohistochemistry (IHC) allows for the detection of IL1RN in tissue sections, particularly useful for studying expression patterns in diseased tissues . Additionally, IL1RN antibodies can be employed in immunoprecipitation (IP) to isolate IL1RN and its binding partners, and in flow cytometry (FACS) to analyze IL1RN expression at the single-cell level . Functionally, neutralizing anti-IL1RN antibodies are valuable tools to block IL1RN activity in cell-based assays, as demonstrated in studies showing reversal of IL1RN-mediated inhibition of IL-1α-dependent cell proliferation .

What is the specificity and cross-reactivity profile of IL1RN antibodies?

The specificity and cross-reactivity of IL1RN antibodies vary significantly between products and should be carefully considered when designing experiments. Most commercially available anti-IL1RN antibodies, such as ABIN5706884, are designed to detect human IL1RN specifically . Cross-reactivity testing reveals that many antibodies raised against human IL1RN also recognize primate IL1RN due to high sequence homology, but typically do not cross-react with mouse or rabbit IL1RN . This species specificity is crucial to consider when planning experiments with animal models. Some antibodies may cross-react with other members of the IL-1 family due to structural similarities, potentially leading to false positive results if not properly validated. Researchers should thoroughly review the validation data provided by manufacturers and consider performing their own validation experiments using appropriate positive and negative controls. For experiments requiring absolute specificity, monoclonal antibodies targeting unique epitopes of IL1RN may be preferable to polyclonal preparations.

How can IL1RN antibodies be used to study inflammatory diseases?

IL1RN antibodies serve as powerful tools for investigating the pathophysiology of inflammatory diseases through multiple approaches. In interstitial lung disease (ILD) research, these antibodies have helped establish that IL1RN expression levels correlate with disease progression and fibrosis development . Immunohistochemical staining with IL1RN antibodies allows researchers to visualize expression patterns in fibro-proliferative areas within affected tissues, as observed in idiopathic pulmonary fibrosis patients . For mechanistic studies, neutralizing anti-IL1RN antibodies can be employed to block endogenous IL1RN function, thereby exacerbating inflammatory responses in experimental systems . This approach has been used to demonstrate that MSC-secreted IL1RN is biologically active and can inhibit IL-1α-dependent cell proliferation, an effect that is abolished when conditioned media is treated with neutralizing anti-IL1RN antibodies . In genetic studies, IL1RN antibodies facilitate the investigation of how polymorphisms or mutations in IL1RN affect protein expression and function, helping to explain why certain variants confer increased risk for inflammatory conditions . Additionally, these antibodies enable researchers to monitor therapeutic responses in experimental models, such as measuring IL1RN levels in bronchoalveolar lavage (BAL) fluid following mesenchymal stem cell (MSC) administration in bleomycin-induced lung injury .

What are the considerations when using IL1RN antibodies in different experimental systems?

When applying IL1RN antibodies across different experimental systems, researchers must consider several system-specific factors that affect antibody performance and data interpretation. In cell culture systems, the choice of cellular model significantly impacts IL1RN expression and detection sensitivity. For instance, MSCs have been identified as high producers of IL1RN, particularly in specific subpopulations, while hematopoietic lineages expressing CD11b, CD34, or CD45 did not show detectable IL1RN protein . Fixed cells for immunocytochemistry require appropriate permeabilization protocols to allow antibody access to intracellular IL1RN, while maintaining the protein's antigenic properties. For tissue sections, the fixation method critically affects epitope preservation and antibody binding; paraformaldehyde fixation may preserve certain epitopes while masking others, necessitating optimization for each antibody . In animal models, the species-specificity of IL1RN antibodies must be thoroughly considered, as many antibodies raised against human IL1RN do not recognize mouse or rabbit IL1RN, despite detecting primate IL1RN . When analyzing biological fluids like bronchoalveolar lavage fluid, researchers must account for potential matrix effects and dilution factors that affect antibody binding and signal detection . In all systems, appropriate controls are essential—positive controls from known IL1RN-expressing samples, negative controls using samples from IL1RN-deficient sources, and isotype controls to address non-specific binding.

How do IL1RN gene mutations affect antibody binding and experimental design?

IL1RN gene mutations can significantly impact antibody binding and necessitate careful consideration in experimental design. The DIRA (deficiency of interleukin-1 receptor antagonist) syndrome, caused by biallelic loss-of-function (LoF) variants in the IL1RN gene, represents a severe monogenic autoinflammatory disease where IL1RN protein is either absent or non-functional . When studying such mutations, researchers must select antibodies targeting epitopes that remain intact in the mutant protein to avoid false negative results. For instance, compound heterozygous IL1RN mutations may affect different domains of the protein, requiring antibodies targeting conserved regions for detection . Epitope mapping becomes crucial when working with patient samples carrying IL1RN mutations—antibodies targeting the specific mutated region will likely fail to bind, while those recognizing preserved regions may still detect the protein but cannot assess its functionality. When analyzing IL1RN expression in carriers of IL1RN mutations (as in family studies), researchers should anticipate altered expression levels due to transcriptional or translational effects . Quantitative analyses comparing wild-type and mutant IL1RN require careful normalization and multiple detection methods, ideally combining protein detection (Western blot, ELISA) with mRNA quantification (qPCR with probes targeting different exons) . In cases where genomic deletions affect the IL1RN locus, specialized PCR approaches may be needed to confirm genetic alterations before attempting protein-level analyses with antibodies .

What are the best practices for quantifying IL1RN using antibody-based methods?

Accurate quantification of IL1RN using antibody-based methods requires rigorous methodology and appropriate controls. For ELISA-based quantification, researchers should generate standard curves using recombinant IL1RN protein of known concentration, ensuring the curve encompasses the expected range of IL1RN in experimental samples . Multiple dilutions of each sample should be tested to confirm linearity and identify potential inhibitory factors in the sample matrix. Western blotting for semi-quantitative analysis requires careful sample preparation with standardized protein loading, verified by housekeeping protein controls such as GAPDH or β-actin . Densitometric analysis of IL1RN bands should be normalized to these loading controls, and multiple biological and technical replicates should be performed to ensure reproducibility. When comparing IL1RN levels between conditions (e.g., control vs. diseased), samples should ideally be processed simultaneously and analyzed on the same blot or ELISA plate to minimize inter-assay variation . For immunohistochemical quantification, consistent staining protocols, objective scoring systems, and computerized image analysis can improve reliability. Researchers studying IL1RN secretion by specific cell populations (such as MSCs) should consider using flow cytometry with intracellular staining to determine the percentage of IL1RN-expressing cells, complemented by ELISA measurements of secreted IL1RN in conditioned media . This combined approach provides insights into both the proportion of IL1RN-producing cells and their secretory capacity.

What are the optimal protocols for using IL1RN antibodies in Western blotting?

Optimizing Western blotting protocols for IL1RN detection requires attention to several critical parameters. Sample preparation should begin with efficient cell or tissue lysis using buffers containing protease inhibitors to prevent IL1RN degradation. For secreted IL1RN, culture media may need concentration via ultrafiltration or immunoprecipitation before analysis . Protein separation is typically performed using 12-15% SDS-PAGE gels to appropriately resolve IL1RN, which has multiple isoforms ranging from 17-25 kDa depending on post-translational modifications. During transfer to membranes, PVDF is often preferred over nitrocellulose due to its higher protein binding capacity and mechanical strength. Blocking should utilize 5% non-fat dry milk or BSA in TBS-T for 1-2 hours at room temperature to minimize non-specific antibody binding . Primary IL1RN antibody incubation conditions vary by product, but typically involve dilutions of 1:500 to 1:2000 in blocking buffer, incubated overnight at 4°C with gentle agitation . After thorough washing with TBS-T (at least 3 x 10 minutes), appropriate HRP-conjugated secondary antibodies should be applied at 1:5000 to 1:10000 dilutions for 1-2 hours at room temperature. Enhanced chemiluminescence detection systems offer suitable sensitivity for most IL1RN detection applications. Critical controls should include positive controls (recombinant IL1RN or lysates from cells known to express IL1RN, such as enriched MSCs), negative controls (samples lacking IL1RN expression), and loading controls (housekeeping proteins) .

How should samples be prepared for optimal IL1RN detection?

Sample preparation methodologies significantly impact IL1RN detection sensitivity and specificity across different experimental applications. For cell culture samples, timing of collection is critical as IL1RN secretion shows temporal dynamics—studies indicate a significant delay (up to 72 hours) between IL-1 stimulation and peak IL1RN secretion . Researchers should consider this delay when designing time-course experiments. For protein extraction from cells or tissues, lysis buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) effectively solubilize IL1RN while preserving its antigenic properties. The addition of protease inhibitor cocktails is essential to prevent degradation during processing . In immunohistochemistry applications, fixation with 4% paraformaldehyde followed by paraffin embedding preserves tissue architecture while maintaining IL1RN antigenicity, though some epitopes may require antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-100°C . For RNA analysis of IL1RN expression, specialized collection tubes like TempusTM effectively stabilize RNA for downstream applications like RT-qPCR . When analyzing biological fluids such as bronchoalveolar lavage (BAL) fluid, samples should be processed promptly and stored with protease inhibitors, as IL1RN can undergo degradation during storage . Centrifugation to remove cellular debris (300-500 x g for 10 minutes) followed by concentration of supernatant may be necessary for samples with low IL1RN concentrations. For genomic analysis, standard DNA extraction methods yield suitable material for PCR amplification and sequencing of IL1RN variants, as demonstrated in studies of IL1RN mutations .

What controls should be used when working with IL1RN antibodies?

Implementing appropriate controls is essential for valid and interpretable results when working with IL1RN antibodies. Positive controls should include recombinant human IL1RN protein at known concentrations or samples from cell types with established IL1RN expression, such as the IL1RN-expressing MSC subpopulation identified in research studies . These controls verify antibody functionality and provide reference signals for comparison. Negative controls should incorporate samples lacking IL1RN expression, such as the CD11b+, CD45+, or CD34+ hematopoietic lineages that have been shown not to express detectable IL1RN protein . For immunostaining applications, isotype controls (primary antibodies of the same isotype but irrelevant specificity) help distinguish specific staining from Fc receptor-mediated or other non-specific binding. Competitive inhibition controls, where excess recombinant IL1RN is pre-incubated with the antibody before sample application, can confirm binding specificity . In functional studies, neutralizing anti-IL1RN antibodies serve as important controls to confirm that observed effects are specifically due to IL1RN activity. For instance, the inhibitory effect of MSC-conditioned media on IL-1α-induced cell proliferation was abolished when treated with neutralizing anti-IL1RN antibody, confirming IL1RN as the principal IL-1α antagonist secreted by murine MSCs . In gene expression studies, multiple TaqMan probes targeting different exons of IL1RN (such as exon 1 and the exon 3-4 junction) provide verification of results and can detect alternatively spliced variants . When quantifying IL1RN expression by qPCR, normalization against at least two endogenous controls (e.g., GAPDH and ACTB) is recommended for reliable results .

How can IL1RN antibodies be used in functional blocking studies?

IL1RN antibodies can be strategically employed in functional blocking studies to elucidate the biological roles of IL1RN in various physiological and pathological contexts. Neutralizing anti-IL1RN antibodies are particularly valuable for these applications, as they can bind to and inhibit the function of IL1RN, effectively restoring IL-1 signaling in experimental systems . Experimental design typically involves pre-incubating samples containing IL1RN (such as conditioned media from IL1RN-expressing cells) with neutralizing antibodies before applying them to test systems. The efficacy of neutralization should be titrated by testing multiple antibody concentrations against a fixed amount of IL1RN, as demonstrated in studies where recombinant IL1RN protein inhibition of D10.G4.1 cell proliferation was blocked by neutralizing anti-IL1RN antibody in a dose-dependent manner . Cell-based assays provide powerful tools for these studies—the IL-1α-dependent helper T lymphocyte cell line D10.G4.1 has been successfully used to assess IL1RN biological activity, as its proliferation is inhibited by IL1RN . Using this system, researchers demonstrated that MSC-conditioned media inhibited IL-1α-induced cell proliferation in a dose-dependent manner, an effect that was abolished by neutralizing anti-IL1RN antibody treatment . When designing blocking experiments, appropriate controls must include isotype control antibodies to account for non-specific effects, along with positive controls (recombinant IL1RN with and without neutralizing antibody) and negative controls (samples lacking IL1RN). For in vivo applications, neutralizing antibodies can be administered systemically or locally to specific tissues to block endogenous IL1RN function and assess its role in disease models, though careful dosing and timing considerations are essential.

How can IL1RN antibodies contribute to understanding DIRA and other IL1RN-related disorders?

IL1RN antibodies provide critical tools for investigating the molecular pathogenesis of DIRA (deficiency of interleukin-1 receptor antagonist) and other IL1RN-related disorders. In clinical diagnostics, these antibodies can be used to screen for IL1RN protein expression in patient samples, helping to distinguish DIRA from other autoinflammatory conditions with similar presentations . When biallelic loss-of-function variants in IL1RN are suspected, immunoblotting with antibodies targeting different epitopes can verify the absence of functional protein and complement genetic testing . For compound heterozygous mutations, as identified in recent case studies, antibodies recognizing distinct domains of IL1RN can help determine which protein regions remain intact and which are affected by each mutation . This epitope-specific approach provides insight into structure-function relationships of IL1RN variants. In family studies, IL1RN antibodies facilitate the analysis of protein expression in carriers of heterozygous IL1RN mutations, revealing how these mutations affect protein production through mechanisms such as nonsense-mediated decay or altered protein stability . For research purposes, IL1RN antibodies enable the characterization of novel therapeutic approaches targeting the IL-1 pathway, including monitoring treatment responses to recombinant IL1RN (anakinra) administration . Additionally, these antibodies support the development of patient-specific in vitro models using technologies like induced pluripotent stem cells, where IL1RN expression and secretion can be assessed in cells derived from patients with IL1RN variants and compared to controls. This approach may reveal cell type-specific effects of IL1RN mutations that contribute to the tissue-specific manifestations observed in DIRA patients.

What role do IL1RN antibodies play in studying mesenchymal stem cell therapy for inflammatory diseases?

IL1RN antibodies have become instrumental in elucidating the therapeutic mechanisms of mesenchymal stem cells (MSCs) in inflammatory diseases. Research has identified a specific subpopulation of MSCs that secrete high levels of IL1RN, which appears to be a key mediator of their anti-inflammatory effects . In characterizing MSC therapies, IL1RN antibodies enable the identification and quantification of IL1RN-producing MSC subpopulations through flow cytometry and immunocytochemistry, with studies revealing that approximately 24% of immunodepleted murine MSCs express IL1RN . This finding has significant implications for cell therapy development, suggesting that enrichment for IL1RN-expressing MSCs could enhance therapeutic efficacy. Functional studies utilizing neutralizing IL1RN antibodies have demonstrated that MSC-secreted IL1RN is biologically active and capable of inhibiting IL-1α function, providing mechanistic insight into how MSCs modulate inflammatory responses . In animal models of lung injury, IL1RN antibodies have helped track changes in IL1RN levels following MSC administration, revealing that MSC treatment inhibits bleomycin-induced increases in both IL1RN mRNA in lung tissue and protein in bronchoalveolar lavage fluid . This counter-intuitive finding suggests that MSCs protect lung tissue by preventing inflammatory responses upstream rather than by simply increasing IL1RN levels . For translational applications, these insights guide the development of potency assays for MSC products, where IL1RN secretion could serve as a biomarker for therapeutic efficacy. The discovery of human MSC subpopulations that express IL1RN provides a strong rationale for developing MSC-based therapies specifically for inflammatory lung diseases and potentially other IL-1-mediated conditions .

How can researchers address common challenges in IL1RN antibody applications?

Researchers frequently encounter several challenges when working with IL1RN antibodies, but methodological refinements can address these issues. For weak or absent signal in Western blotting, increasing protein loading (up to 50-100 μg per lane), extending primary antibody incubation time (overnight at 4°C), optimizing antibody concentration, and employing more sensitive detection systems like enhanced chemiluminescence plus (ECL+) can improve results . Background issues in immunostaining can be minimized by extending blocking time (2-3 hours), using alternative blocking agents (2-5% BSA instead of serum), increasing wash duration and frequency, and titrating primary and secondary antibodies to optimal concentrations . Cross-reactivity problems may be addressed by pre-absorbing antibodies with related proteins or by selecting more specific monoclonal alternatives targeting unique epitopes of IL1RN . Inconsistent results between experiments often stem from variations in sample preparation or storage conditions—standardizing these protocols and including internal controls in each experiment improves reproducibility . For detecting secreted IL1RN in culture media, protein loss during processing can be minimized by adding carrier proteins (0.1% BSA) to dilute samples and using low-protein-binding tubes and filters . When quantifying IL1RN by ELISA, matrix effects from complex biological samples can interfere with antibody binding—these can be mitigated by diluting samples in assay buffer, using calibrators prepared in similar matrices, or employing sample pre-treatment steps to remove interfering components . For functional blocking studies, incomplete neutralization may occur if antibody concentration is insufficient—performing antibody titration experiments and extending pre-incubation time (2-3 hours) before adding to experimental systems increases blocking efficacy .

How can researchers validate the specificity of IL1RN antibodies?

Comprehensive validation of IL1RN antibody specificity is essential for generating reliable research data. Researchers should implement multiple complementary approaches to confirm antibody specificity. Western blotting with recombinant IL1RN protein serves as a primary validation method, confirming the antibody detects a protein of the expected molecular weight (approximately 17-25 kDa depending on isoform and post-translational modifications) . Parallel analysis of samples from sources known to express IL1RN (such as the MSC subpopulation) and those lacking IL1RN expression (like CD11b+/CD45+ hematopoietic cells) provides biological validation of specificity . Competitive inhibition assays, where pre-incubation of the antibody with excess recombinant IL1RN blocks subsequent binding to endogenous IL1RN in samples, offer further confirmation of specific binding . RNA interference (siRNA or shRNA) to knockdown IL1RN expression followed by immunoblotting should show corresponding reduction in antibody-detected signal if the antibody is specific. For anti-IL1RN antibodies intended for neutralization studies, functional validation is critical—researchers should demonstrate that the antibody blocks the biological activity of IL1RN, as shown in studies where neutralizing antibodies reversed the inhibitory effect of IL1RN on IL-1α-dependent cell proliferation . Cross-reactivity assessment with other IL-1 family members helps establish specificity within this structurally related protein family. When analyzing patient samples with known IL1RN mutations, antibodies targeting different epitopes can be used to verify specificity—those targeting regions affected by mutations should show altered binding patterns compared to antibodies recognizing preserved regions . For absolute confirmation of specificity, mass spectrometry analysis of immunoprecipitated proteins can identify whether the antibody-captured protein is indeed IL1RN and not a cross-reactive species.