Standard curve
Example of Human IL-33 standard curve in Assay Diluent K1.
Human IL-33 culture media OneStep ELISA Kit
Human IL-33 culture media OneStep ELISA Kit
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Product Details
Product Details
Product Specification
| Antigen | IL-33 |
| 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: 3.2% Inter-assay: 3.9% |
| Sample type | Cell culture supernatant |
| Assay type | Sandwich (quantitative) |
| Sensitivity | 1.36 pg/mL |
| Range | 11.7 pg/mL – 750 pg/mL |
| Recovery | Cell culture supernatant (1640): 87% Cell culture supernatant (DMEM): 92% |
| Assay time | 60 minutes |
Background
Human interleukin-33 (IL-33), also known as IL-1 family member 11 (IL-1F11) or nuclear factor from high endothelial venules (NF-HEV), is a dual-function cytokine belonging to the IL-1 superfamily. Under homeostatic conditions, the full-length protein (270 amino acids in the canonical human sequence) is constitutively expressed in the nucleus of barrier epithelial cells and endothelial cells, where it acts as a chromatin-associated transcriptional repressor. Upon cellular damage or necrosis, IL-33 is released into the extracellular space and functions as an "alarmin" that signals through the ST2 (IL1RL1) receptor complex, activating NF-κB and MAPK pathways to drive type 2 immune responses. Its biological activities include promoting Th2 cell maturation, activating mast cells, basophils, eosinophils, and NK cells, and inducing the production of IL-4, IL-5, and IL-13. The mature, biologically active form of human IL-33 is generated by proteolytic cleavage (e.g., by caspase-1) and is commonly produced recombinantly in E. coli as a non-glycosylated polypeptide of approximately 159–160 amino acids with a molecular weight of about 17.9–18.1 kDa. Given its central role in allergic inflammation, tissue repair, and tumor immunity, IL-33 is an important research target and a potential therapeutic candidate.
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ELISA
Spike Recovery
The recovery of Human IL-33 was evaluated in activated samples spiked with concentrations spanning the entire assay range.
Spike-and-dilution Linearity
To evaluate assay linearity, the sample was spiked with high levels of Human IL-33 in cell culture media and serially diluted with the corresponding Calibrator Diluent to fall within the assay's dynamic range.
Cross-reactivity
The cross-reactivity between Mouse IL-33 and Human IL-33 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-33 was tested in the Human IL-33 assay, as the signals remained within the background range of the assay.
Determination of Minimum Detectable Dose (MDD)
The MDD was determined using three independent lots of assay components. For each lot, 19 replicate measurements of the diluent (zero calibrator) were performed. The mean (AVERAGE) and standard deviation (STDEV) of the 19 replicates were calculated. The MDD for each lot was then calculated according to the following formula:
MDD = 2 × STDEV + AVERAGE
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.
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.
Dilution Linearity
The concentrations of Human IL-33 were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph represents neat biological samples. "Undiluted" was defined as the neat biological sample (100%). The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 36.1 pg/mL in the stimulated THP-1 cell supernatant.
Protocol Diagram
