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ELISA OD to Concentration Calculator

Reviewed by the CalculatorHub team, edited by James Graham. Log-log linear regression of standard curve. Last verified 14 June 2026.

ELISA (enzyme-linked immunosorbent assay) quantifies analyte concentration by comparing sample optical density (OD) values to a standard curve of known concentrations. This calculator builds a standard curve using log-log linear regression of your standard OD and concentration values, then interpolates unknown sample concentrations from their OD readings. Enter the standard concentrations and ODs (comma-separated, in matching order), then enter unknown sample ODs to get their concentrations.

Known concentrations of your standards
Measured OD for each standard (same order as concentrations)
OD reading(s) of the sample(s) to quantify
Multiply result by this if sample was diluted before assay
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Standard curve method

log10(OD) = a + b * log10(concentration)

log10(concentration) = (log10(OD) - a) / b

The calculator fits a linear regression on log10(OD) vs log10(concentration) for the standard curve points, then inverts the relationship to estimate concentration from an unknown OD. This approach approximates the linear dynamic range of a typical ELISA. For a full 4-parameter logistic model, use dedicated ELISA analysis software.

ELISA quantification best practices

  • Run standards in duplicate or triplicate and average replicates before curve fitting.
  • Include a blank (diluent only, no analyte) well and subtract from all readings.
  • Dilute samples that fall above the highest standard to bring them within the curve range.
  • Report values outside the linear range as greater than the highest standard or less than the lowest standard, not as interpolated values.
  • Check R-squared; values above 0.99 indicate a good standard curve fit.

ELISA concentration calculation: frequently asked questions

What is an ELISA standard curve?

An ELISA standard curve is a graph of known analyte concentrations (x-axis) versus measured optical density or OD (y-axis) values from the plate reader. Unknown sample concentrations are determined by finding where their OD falls on this curve.

Why is log-log regression used for ELISA standard curves?

ELISA signals are typically sigmoidal (S-shaped) across a wide concentration range. However, within the dynamic linear range of the assay, a log-log plot of concentration vs OD is approximately linear, allowing simple interpolation. For a full sigmoidal fit, a 4-parameter logistic (4PL) model is preferred.

What is a good OD range for accurate ELISA quantification?

Accurate quantification is generally possible within the linear range of the standard curve, where OD values lie between approximately 0.1 and 2.5. Samples with OD above the highest standard should be diluted and re-assayed.

What wavelength is typically used for ELISA?

Most colourimetric ELISAs using TMB (3,3',5,5'-tetramethylbenzidine) substrate are read at 450 nm with a reference wavelength of 620 or 650 nm. HRP with OPD substrate is read at 490 nm. The specific wavelength depends on the substrate used.

How do I account for sample dilution in ELISA?

The final concentration in the original sample = interpolated concentration * dilution factor. If you diluted 1:10 before running the ELISA and the interpolated value is 5 ng/mL, the concentration in the original sample is 50 ng/mL.

Official sources

Reviewed by the CalculatorHub team, edited by James Graham, 14 June 2026. See our methodology.