Standard Curve
Example of Human IL-6 standard curve in Assay Diluent FE6. Plotted are the background-subtracted data.
This standard curve is provided for demonstration only.
Human IL-6 OneStep ELISA Kit
Human IL-6 OneStep ELISA Kit
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Product Details
Product Details
Product Specification
| Antigen | IL-6 |
| Immunogen | Recombinant Protein |
| Antibody Type | Recombinant mAb |
| Reactivity | Hu |
| Purification | Protein A |
| Stability & Storage | 12 months from date of receipt / reconstitution, 2 to 8°C as supplied. |
Kit
| Precision | Intra-assay: 2.9% Inter-assay: 4.5% |
| Sample type | Cell culture supernatant; Serum; Plasma |
| Assay type | Sandwich (quantitative) |
| Sensitivity | 9.15 pg/mL |
| Range | 31.25 pg/mL – 2000 pg/mL |
| Recovery | Cell culture supernatant: 106% Human Serum: 105% Heparin Plasma: 108% EDTA Plasma: 116% Citrate Plasma: 111% |
| Assay time | 60 minutes |
| Species reactivity | Hu |
Background
Human Interleukin-6 (IL-6) is a multifunctional cytokine produced by various cell types, including T cells, macrophages, fibroblasts, and endothelial cells. It plays a central role in regulating the immune response, inflammation, and hematopoiesis. IL-6 exerts its effects by binding to a receptor complex consisting of the IL-6 receptor alpha (IL-6Rα) and the signal-transducing subunit gp130. Upon binding, it activates downstream signaling pathways such as JAK/STAT, MAPK, and PI3K. While acute IL-6 expression is protective during infection or tissue injury, chronic dysregulation of IL-6 is associated with numerous pathological conditions, including rheumatoid arthritis, Castleman’s disease, certain cancers, and cytokine release syndrome (e.g., in severe COVID-19). As a result, IL-6 and its receptor are important therapeutic targets, with agents like tocilizumab (anti-IL-6R) being used clinically to manage inflammatory diseases.
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ELISA
Spike Recovery
The recovery of Human IL-6 was evaluated in activated samples spiked with concentrations spanning the entire assay range.
Dilution Linearity
The concentrations of Human IL-6 were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph shows biological samples diluted in Assay Diluent to 6.3% concentration. "undiluted" was defined as 6.3% biological sample. The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 49.443 ng/mL in human PBMC culture supernatant stimulated with 10 ug/mL PHA for 5 days.
Dilution Linearity
The concentrations of Human IL-6 were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph shows biological samples diluted in Assay Diluent to 100% concentration. "undiluted" was defined as 100% biological sample. The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 72.75 pg/mL in human PBMC culture supernatant.
To assess the linearity of the assay, the sample was spiked with high concentrations of Mouse IL-4R in various matrices and diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested fifteen times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays)
Three samples of known concentration were tested in separate assays to assess inter-assay precision. Assays were performed with at least three lots of components.
Determination of Minimum Detectable Dose (MDD)
The MDD was determined using three independent lots of assay components. For each lot, 20 replicate measurements of the diluent (zero calibrator) were performed. The mean (AVERAGE) and standard deviation (STDEV) of the 20 replicates were calculated. The MDD for each lot was then calculated according to the following formula:
MDD = 2 × STDEV + AVERAGE
Cross-reactivity
The cross-reactivity between Human IL-6 and Mouse IL-6 was assessed by testing both proteins in the same assay system. Serial dilutions of each protein were measured, and the dose-response curves were compared. No significant cross-reactivity was observed when Mouse IL-6 was tested in the Human IL-6 assay, as the signals remained within the background range of the assay.
HOOK Effect Threshold
The upper limit of the HOOK effect was established at 100× the highest calibrator concentration (equivalent to a 2-log10 increase). No HOOK effect was observed below this threshold, confirming that samples within this concentration range yield reliable quantitative results without signal depression.
Protocol Diagram
