sRANKL Human, His
Description
Biological Functions and Mechanisms
sRANKL is a homotrimeric cytokine critical for osteoclast differentiation and immune cell interactions:
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Osteoclastogenesis: Binds RANK (TNFRSF11A) to activate NF-κB and MAPK pathways, driving bone resorption .
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Immune Regulation: Enhances dendritic cell survival and T-cell proliferation, modulating adaptive immunity .
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Decoy Receptor Interaction: Neutralized by osteoprotegerin (OPG), a soluble inhibitor that prevents excessive bone loss .
Clinical Relevance:
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Bone Diseases: Elevated sRANKL levels correlate with osteoporosis, hypercalcemia, and metastatic bone lesions .
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Metabolic Syndromes: Low serum sRANKL associates with obesity, insulin resistance, and cardiovascular risk .
Table 1: sRANKL in Metabolic and Critical Illness Studies
Table 2: Therapeutic Targeting of sRANKL
Applications in Biomedical Research
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Osteoclast Differentiation Assays: Used to model bone resorption in vitro .
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Immune Studies: Evaluates dendritic cell-T cell interactions in autoimmune diseases .
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Drug Development: Screens for osteoporosis therapies targeting sRANKL-RANK binding .
Challenges and Future Directions
Product Specs
Soluble Receptor Activator of NFkB Ligand, TNFSF11, TRANCE, TNF-related activation-induced cytokine, OPGL, ODF, Osteoclast differentiation factor, Tumor necrosis factor ligand superfamily member 11, Receptor activator of nuclear factor kappa B ligand, RANKL, Osteoprotegerin ligand, CD254 antigen, sRANKL, sOdf, hRANKL2.
MGSSHHHHHH SSGLVPRGSH MIRAEKAMVD GSWLDLAKRS KLEAQPFAHL TINATDIPSG SHKVSLSSWY HDRGWAKISN MTFSNGKLIV NQDGFYYLYA NICFRHHETS GDLATEYLQL MVYVTKTSIK IPSSHTLMKG GSTKYWSGNS EFHFYSINVG GFFKLRSGEE ISIEVSNPSL LDPDQDATYF GAFKVRDID.
Q&A
What is sRANKL and what are its standard reference ranges in healthy human subjects?
sRANKL (soluble Receptor Activator of Nuclear Factor Kappa-B Ligand) is a tumor necrosis factor that binds to RANK and Osteoprotegerin (OPG). It activates the AKT pathway and mediates osteoclastogenesis and T cell activation . As a member of the TNF superfamily (TNFSF11), sRANKL plays critical roles in bone metabolism and immune function.
In terms of reference ranges, screening of 32 healthy human donors revealed the following values for free soluble RANKL in serum:
| Statistical Parameter | Value (pmol/l) |
|---|---|
| Mean | 0.17 |
| Median | 0.14 |
| 95th Percentile | 0.43 |
| 5th Percentile | 0.04 |
It's important to note that laboratory-specific reference ranges should be established, as these values can vary between testing facilities . The median value (0.14 pmol/l) falls between calibration points 3 and 4 of a standard curve in high-sensitivity ELISA systems .
How do ELISA-based methods for sRANKL detection compare in sensitivity and what are their detection limits?
High-sensitivity sandwich ELISA methods have become the standard for sRANKL detection in research applications. Current generation assays offer significant advantages in detecting physiological levels of free sRANKL.
The typical performance characteristics of high-sensitivity sRANKL ELISA include:
| Parameter | Value |
|---|---|
| Detection limit | 0.01 pmol/l (0.2 pg/ml) |
| Lower limit of quantification | 0.008 pmol/l |
| Standard range | 0-2 pmol/l (0-40 pg/ml) |
| Conversion factor | 1 pmol/l = 20 pg/ml (MW: 20 kDa) |
| Sample volume required | 150 μl/well |
| Precision (in-between-run) | ≤3% CV |
| Precision (within-run) | ≤5% CV |
The newest generation of assays can detect free, uncomplexed soluble RANKL, which is crucial for accurately measuring the biologically active form rather than RANKL bound to OPG . This specificity is particularly important when studying conditions where the balance between sRANKL and OPG may be altered.
What are the critical considerations for sample collection and processing when measuring sRANKL levels?
Proper sample collection and processing are essential for accurate sRANKL quantification. Research-grade protocols should adhere to the following guidelines:
Acceptable sample types include serum and heparin plasma . Sample stability studies indicate that proper storage is critical - samples should be processed promptly and stored at -80°C for long-term preservation to prevent degradation of the target protein.
The standard assay procedure typically involves:
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Pre-washing wells coated with recombinant OPG
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Adding assay buffer to microtiter wells
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Adding 150 μl of standard/control/sample per well
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Incubation allowing sRANKL binding to pre-coated OPG
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Washing to remove unbound material
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Addition of biotinylated detection antibody (typically polyclonal goat anti-human sRANKL)
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Detection through standard ELISA visualization methods (HRP/TMB)
The timing protocol generally follows: 2 hours initial incubation, overnight second incubation, 1 hour third incubation, and 30 minutes final development .
How should researchers address the challenge of undetectable sRANKL levels in statistical analyses?
The issue of undetectable levels presents a common analytical challenge in sRANKL research. Based on statistical approaches employed in published studies, researchers should consider the following methodological solutions:
For samples with undetectable levels (below the lower detection limit of 0.001 pmol/l), statistical handling involves coding these values at 0.0005 pmol/l, representing the midpoint between 0 and the lower detection limit . This approach prevents the exclusion of potentially important data points.
When analyzing data with a significant proportion of undetectable values, appropriate statistical transformations should be employed:
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Log transformation of sRANKL values (log10 scale) when measurements vary by orders of magnitude
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Quartile-based analysis approaches, where undetectable levels can define the lowest quartile
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Non-parametric statistical methods when distribution assumptions are not met
In published research, quartile cutpoints are typically defined based on the distribution in control groups, with appropriate adjustments in logistic regression models .
How can sRANKL and sRANKL/OPG ratio measurements be optimized for biomarker research in oncology?
The application of sRANKL and sRANKL/OPG ratio as biomarkers in oncology represents an advanced research domain with significant potential. Based on studies in non-small cell lung cancer (NSCLC), the following methodological considerations should be incorporated:
ROC curve analysis has established optimal diagnostic cut-off points for sRANKL level and sRANKL/OPG ratio at 4.20 pmol/l and 0.60, respectively, for distinguishing NSCLC patients from those with benign conditions or healthy controls . These thresholds demonstrated the following performance characteristics:
| Biomarker | AUC (95% CI) | Sensitivity | Specificity | Accuracy |
|---|---|---|---|---|
| sRANKL level | 0.837 (0.746-0.927) | 74.0% | 84.0% | 77.3% |
| sRANKL/OPG ratio | 0.922 (0.863-0.980) | 84.0% | 88.0% | 85.3% |
For differentiating between benign and malignant pulmonary lesions specifically, the optimal cut-off points shift to 5.24 pmol/l for sRANKL and 0.63 for sRANKL/OPG ratio . This refinement in diagnostic thresholds highlights the importance of context-specific calibration in biomarker applications.
Researchers should note that the sRANKL/OPG ratio consistently outperforms sRANKL levels alone in diagnostic performance, suggesting the importance of measuring both markers simultaneously .
What spike recovery protocols should be implemented to validate sRANKL assay performance with endogenous samples?
Spike recovery experiments are essential for validating assay performance with endogenous free soluble RANKL. Based on established validation protocols, researchers should implement the following approach:
For matrix evaluation, mixing experiments should be conducted using samples with known endogenous sRANKL concentrations. Validation data suggests the following recovery rates can be expected when mixing samples in different proportions:
| Matrix Type | Mixing Ratio 1+1 | Mixing Ratio 1+4 |
|---|---|---|
| Serum | 89% (0.15 pmol/l) | 85% (0.15 pmol/l) |
| Heparin plasma | 100% (0.15 pmol/l) | 98% (0.15 pmol/l) |
These recovery rates indicate that heparin plasma may provide marginally better recovery performance than serum in spike recovery experiments . Researchers should consider these matrix effects when selecting sample types for their specific experimental questions.
For cases where samples containing higher endogenous levels of free soluble RANKL are not available, specialized experimental designs may be required, as noted in assay validation documentation .
What quality control measures should be implemented when working with sRANKL ELISA systems?
Robust quality control is critical for obtaining reliable and reproducible sRANKL measurements. The following comprehensive QC protocol is recommended based on established research practices:
Standard curve validation should employ a 4-parameter logistic (4PL) curve fitting model, which provides optimal modeling of the typical sRANKL ELISA response curve . The standard curve should include 7 calibration points ranging from 0 to 2 pmol/l (specifically: 0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 pmol/l) plus two serum-based controls .
Inter-assay variation should be monitored and maintained below 3% CV (coefficient of variation), while intra-assay variation should remain below 5% CV . Exceeding these thresholds suggests technical issues requiring investigation.
How can researchers address potential confounding factors when interpreting sRANKL results?
Interpretation of sRANKL data requires careful consideration of potential confounding factors. Based on correlation studies, the following factors should be evaluated:
Age represents a significant confounding factor for OPG levels (correlation coefficient r = 0.33), with mean age ranging from 67.7 years in the lowest quartile (≤4 pmol/L) to 72.6 years in the highest quartile (>6 pmol/L) . Interestingly, sRANKL levels were not significantly associated with age in control populations.
Additional factors showing significant associations with OPG levels include:
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Treated diabetes status
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Self-rated health status
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Physical function scores
For sRANKL specifically, total calcium intake showed significant associations, with notably lower intake observed in the lowest quartile of sRANKL (0.0005 pmol/L) .
Hormone therapy (HT) status does not appear to significantly affect sRANKL or OPG levels across quartiles, though this should be considered in specific research contexts .
To address these potential confounders, statistical approaches should include:
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Spearman correlations to assess associations between biomarkers and continuous covariates
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Adjustment for matching factors in regression models (age, sample collection timing)
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Stratified analyses for factors like hormone therapy status, body mass index, and FRAX score
What are the emerging applications of sRANKL measurement in interdisciplinary research?
The utility of sRANKL extends beyond traditional bone metabolism research into multiple interdisciplinary areas. Emerging research directions include:
In oncology, sRANKL and sRANKL/OPG ratio have demonstrated superior performance compared to traditional cancer biomarkers like CEA and CA19-9, particularly for lung cancer detection . This suggests potential applications in early cancer detection protocols, especially for patients with atypical imaging patterns or small tumor diameters (<2.5 cm).
In fracture risk assessment, investigations into associations between serum levels of OPG, sRANKL, and the OPG/sRANKL ratio and incident hip fracture risk suggest potential applications in osteoporosis management . These biomarkers may complement existing fracture risk assessment tools by providing insights into underlying bone turnover mechanisms.
Future research should focus on large-scale validation studies comparing sRANKL and sRANKL/OPG ratio against currently used clinical markers, establishing standardized reference ranges across diverse populations, and investigating the biological mechanisms underlying observed associations .
How can researchers effectively combine sRANKL with other biomarkers for comprehensive pathway analysis?
Multimarker approaches incorporating sRANKL offer enhanced research capabilities. Based on current literature, researchers should consider:
Combining sRANKL measurements with other bone turnover markers can provide more comprehensive insights into bone metabolism status. Key complementary markers include:
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Osteoprotegerin (OPG) - the natural decoy receptor for RANKL
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Parathyroid hormone (PTH) - though correlation with sRANKL appears limited (r = 0.02)
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Calcium markers - particularly relevant given observed associations with sRANKL levels
For cancer research applications, the combination of sRANKL/OPG ratio with traditional cancer markers shows promise for enhancing diagnostic accuracy. Future studies should explore multi-marker panels incorporating sRANKL with:
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CEA (Carcinoembryonic antigen)
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CA19-9 (Cancer antigen 19-9)
When implementing multimarker approaches, researchers should employ advanced statistical methods including multivariate analyses to account for potential correlations between markers, and consider machine learning approaches for identifying optimal biomarker combinations for specific research questions.
Product Science Overview
The recombinant human sRANKL is typically produced in Escherichia coli (E. coli) and purified through sequential chromatography . The protein is a non-glycosylated polypeptide chain containing 199 amino acids, with a molecular mass of approximately 22.3 kDa . The His tag, consisting of 21 amino acids, is fused to the N-terminus of the protein to facilitate purification and detection .
sRANKL is known for its ability to bind to the receptor activator of nuclear factor kappa-B (RANK) on the surface of osteoclast precursors, promoting their differentiation into mature osteoclasts. This interaction is essential for bone resorption and remodeling . Additionally, sRANKL can interact with a decoy receptor called osteoprotegerin (OPG), which inhibits osteoclastogenesis by preventing sRANKL from binding to RANK .
Recombinant sRANKL is used extensively in cell culture, differentiation studies, and functional assays. It is particularly valuable in research focused on bone diseases, such as osteoporosis and rheumatoid arthritis, as well as in studies of immune system regulation . The protein’s ability to induce NF-κB activation in various cell types makes it a powerful tool for investigating signaling pathways involved in inflammation and immune responses .
The lyophilized form of sRANKL is typically reconstituted in distilled water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to store the reconstituted solution in working aliquots at -80°C to -20°C, with a carrier protein such as 0.1% bovine serum albumin (BSA) to enhance stability . The lyophilized protein is stable at room temperature for up to one month but should be stored at -20°C for extended periods .
