Data analysis · Beta · Free Verbex lab tool
ELISA Standard Curve Calculator (4PL)
Paste your standards and samples straight from the plate reader. Verbex fits a 4PL curve, checks every standard against it, sets the working range and reports sample concentrations only where the curve supports them.
Standard curve: IL-6 ELISA, plate 1
Curve passes: 8 of 8 standards within limits. Working range 4.69–600 pg/mL.
Fit: 4PL, weighted 1/Y², R² 0.9984 (a = 0.03192, b = 1.17, c = 239, d = 3.924). F test p = 0.150: 5PL does not fit significantly better.
Blank: mean 0.0505. Blanks subtracted as background: the signal rises with concentration (sandwich ELISA, BCA, Bradford).
Weighting follows immunoassay guidance; GraphPad Prism and Excel templates fit unweighted by default, which can shift results near the ends of the curve.
| Standard (pg/mL) | Mean response | CV | Back-calculated | Recovery | Result |
|---|---|---|---|---|---|
| 4.69 | 0.0705 | 3.5% | 4.678 | 99.7% | pass |
| 9.38 | 0.1175 | 2.5% | 9.337 | 99.5% | pass |
| 18.75 | 0.225 | 2.8% | 19.18 | 102.3% | pass |
| 37.5 | 0.4245 | 2.1% | 36.86 | 98.3% | pass |
| 75 | 0.8245 | 2.3% | 74.53 | 99.4% | pass |
| 150 | 1.445 | 1.6% | 147.7 | 98.5% | pass |
| 300 | 2.319 | 1.7% | 323.3 | 107.8% | pass |
| 600 | 2.876 | 1.2% | 560.6 | 93.4% | pass |
| Sample | Dilution | Mean response | CV | Concentration (pg/mL) | Result |
|---|---|---|---|---|---|
| Patient 01 | 1:2 | 0.6125 | 1.7% | 108 | in range |
| Patient 02 | 1:2 | 3.014 | 0.7% | > 1200 | above range: dilute further |
| Patient 02 | 1:20 | 1.169 | 2.3% | 2245 | in range |
| Patient 03 | neat | 0.01 | 1.2% | < 4.69 | below range |
Limits: ICH M10 defaults (recovery ±20%, ±25% at LLOQ/ULOQ; replicate CV ≤ 20%; at least 75% and 6 levels passing).
Check: every standard put back through the fitted curve reproduces its mean response ✓
Beta method, checked against published datasets; the choices behind this result are listed with it.
Verbex Lab Tools · Standard curve v0.1.0-beta · 2026-10-15 15:50 UTC · verify before use
- Patient 02 (1:2) reads above the ULOQ (600 pg/mL); dilute it further and rerun.
- Patient 03 reads below the LLOQ (4.69 pg/mL); report as < 4.69 pg/mL, or run it less diluted.
How it is calculated
Every number shows its work.
Verbex fits a four-parameter logistic (4PL) curve to every replicate well, weighted 1/Y² because ELISA signal varies more at high concentrations. A 5PL is used only with at least 7 standards and when an F test shows it fits significantly better.
Each standard is read back through the fitted curve. By default it passes if it recovers within ±20% of its nominal concentration (±25% at the ends) with replicate CV of 20% or less, and the curve passes if at least 75% of standards and 6 levels pass. These limits follow ICH M10; change them under More options, and the result always states which limits it used.
The working range runs from the lowest to the highest passing standard. Samples inside it are read off the curve and multiplied by their dilution factor; samples outside it are reported as below or above range, never extrapolated. Nothing is excluded unless you give a reason.
This is a beta, checked against published data: 7 published datasets (13 curve fits, including GraphPad Prism's example, R's drc and a competitive ELISA plate with the reader's own results) reproduce within 1% with matching settings. Treat results as checked, and compare them with your lab's requirements.
Use it from your AI assistant
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