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ELISA standard curve analyzer (4PL)

Enter your standards (concentration and OD, with replicates) and your samples’ ODs and dilution factors. The analyzer fits a four-parameter logistic (4PL) curve, or a linear or log-log line, subtracts the blank, and calculates each sample’s concentration, flagging values outside the standard range. You get R², the fitted parameters, a chart and a CSV.

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What it does

  • 4PL (recommended), linear and log-log models

  • Replicates with CV %

  • Blank (zero standard) subtraction

  • Dilution factors applied to unknowns

  • Flags samples outside the standard range

  • Chart (PNG/SVG) and CSV

How to use the ELISA standard curve analyzer

  1. Enter the standardsConcentration and up to three OD replicates per standard; paste from a plate-reader export or type them in.
  2. Enter the samplesName, OD replicates and dilution factor for each unknown.
  3. Fit and readKeep the 4PL model unless your kit says otherwise; read the concentrations and check the range flags, then export.

How it works

4PL: y = Bottom + (Top − Bottom) / (1 + (EC50 / x)Hill)
x = EC50 × ((Top − Bottom)/(y − Bottom) − 1)−1/Hill

The 4PL curve is fitted by Levenberg–Marquardt least squares on log concentration (the same fitter as the LabCanvas Graphs app, validated against SciPy), using every replicate as its own point. The mean OD of each unknown, minus the blank, is converted back to a concentration with the inverse of the curve and multiplied by its dilution factor. Values above the top standard or below the lowest are flagged, because extrapolated concentrations are unreliable.

Worked example

With the example data (a 1000 to 15.6 pg/mL series), the blank (zero standard) averages 0.062 and the 4PL fit has R² = 0.9997 and an EC50 of about 280 pg/mL. Sample 1 has a mean OD of 0.947 (0.885 after the blank) and a dilution of 2: the curve reads about 139 pg/mL in the well, so the sample contains about 277 pg/mL.

Frequently asked questions

Which curve fit should I use for ELISA?

The four-parameter logistic (4PL) is the standard for sandwich and competitive ELISAs because it follows the S-shaped response, including the flat ends. Use linear only if the kit specifies it and your standards are in the linear range.

What is a good R² for an ELISA standard curve?

Usually 0.99 or higher for a 4PL fit. Also check that standards back-calculate to within about 80–120 % of their nominal values and that replicate CVs are under about 15–20 %.

What should I do with samples above the top standard?

Dilute them further and re-run them. Concentrations extrapolated beyond the standard range are not reliable.

Should I subtract the blank?

Yes, if the kit includes a zero standard or blank wells; it removes background signal. The analyzer subtracts the mean of the zero standard when the option is on.

How do dilution factors work?

If you diluted a sample 1:10 before the assay, enter 10: the concentration read from the curve is multiplied by 10 to give the original sample’s concentration.

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