Standard Curve
Example of Rat TNF-α standard curve in Assay Diluent A1. Plotted are the background-subtracted data.
This standard curve is provided for demonstration only.
Rat TNF-α Culture media OneStep ELISA Kit
Rat TNF-α Culture media OneStep ELISA Kit
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Product Details
Product Details
Product Specification
| Antigen | TNF-α |
| Immunogen | Recombinant Protein |
| Antibody Type | Recombinant mAb |
| Reactivity | Rt |
| Purification | Protein A |
| Stability & Storage | 12 months from date of receipt / reconstitution, 2 to 8°C as supplied. |
Kit
| Precision | Intra-assay: 1.8%； Inter-assay: 3.2% |
| Sample type | Cell Culture Supernatant |
| Assay type | Sandwich (quantitative) |
| Sensitivity | 2.205 pg/mL |
| Range | 7.81 pg/mL – 500 pg/mL |
| Recovery | Cell Culture Supernatant: 86% |
| Assay time | 60 minutes |
| Species reactivity | Rt |
| CROSS REACTIVITY | Ms |
Background
Rat TNF-α (Tumor Necrosis Factor-alpha) is a multifunctional pro-inflammatory cytokine centrally involved in immune regulation, inflammation, and cell survival, primarily produced by activated macrophages as well as other cell types such as T lymphocytes and fibroblasts; synthesized as a 26 kDa transmembrane precursor and cleaved by TACE into a soluble 17 kDa active homotrimer, it exerts its effects by binding to TNFR1 and TNFR2 receptors on nearly all nucleated cells, thereby activating NF-κB and MAPK signaling pathways to drive diverse outcomes including inflammation, apoptosis, proliferation, and tissue remodeling, and its dual role as both a host defense mediator and a pathological inflammation driver makes it a critical therapeutic target and key biomarker extensively studied in rat models of autoimmune diseases, septic shock, cancer, and chronic inflammatory conditions like rheumatoid arthritis and inflammatory bowel disease.
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ELISA
Spike Recovery
The recovery of Rat TNF-α was evaluated in activated samples spiked with concentrations spanning the entire assay range.
Dilution linearity
The concentrations of Rat TNF-α 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 188.85 pg/mL in Rat spleen tissue culture supernatant stimulated with 100 ng/mL LPS for 3 Days.
Dilution linearity
The concentrations of Rat TNF-α 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 40.12 pg/mL in Rat spleen tissue culture unstimulated supernatant.
Spike-and-dilution Linearity
To evaluate assay linearity, the sample was spiked with high levels of Rat TNF-α in different matrices and serially diluted with the corresponding Calibrator Diluent to fall within the assay's dynamic range.
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, 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
Cross‑reactivity
The cross‑reactivity between Mouse TNF-α、Human TNF-α and Monkey TNF-α 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 Human TNF-α and Monkey TNF-α was tested in the Rat TNF-α 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
