Why fit a 4PL curve instead of a straight line?

ELISA signal usually flattens at very low and very high concentrations, so a straight line only matches the middle. A four-parameter logistic curve estimates the lower plateau, upper plateau, midpoint, and slope.

Why is there no number for a sample outside the standard range?

Outside the standards the curve is no longer reliable, so this tool does not extrapolate. Dilute a sample whose concentration is above the range and measure it again, then multiply by the dilution factor. A sample below the lowest standard is reported only as less than that standard times its dilution.

Why is the zero standard a blank in a sandwich assay and B0 in a competitive assay?

In a sandwich or indirect assay the zero standard is the blank and is subtracted from every well. In a competitive assay the zero standard is B0, the maximum signal, and is not subtracted.

Research workbench

ELISA Standard Curve

Fit a 4PL standard curve and back-calculate sample concentrations

Data is processed in this browser and is never uploaded.

The zero standard is the blank and is subtracted from every well.

Replicates

Standards
Concentration	OD 1	OD 2	Remove

Samples
Name	OD 1	OD 2	Dilution	Remove

Enter standard and sample ODs to see the curve and back-calculated concentrations.

How to read an ELISA standard curve

Enter the standard concentrations and their replicate ODs, then the unknown samples with the dilution used on the plate. The curve is fitted in this browser. A zero standard is treated differently for the two formats described below.

Why 4PL instead of a straight line

At the low end the signal sits on the baseline. At the high end the reagents are saturated and extra analyte barely changes the OD. A straight line cannot describe both plateaus. 4PL follows that sigmoid shape, which is why the midpoint (EC50) is reported with R².

Out of range means dilute and measure again

If a sample's concentration is beyond the highest standard, or below the lowest, the tool withholds a number. Do not extend the curve by eye. Dilute a high sample, measure it again, and multiply by that dilution. A low sample is shown only as less than the lowest standard times its dilution. In a competitive assay OD falls as concentration rises, so a sample above the range reads lower than the highest standard.

Sandwich blank versus competitive B0

For a sandwich or indirect assay, the mean OD of the zero standard is the blank subtracted from the plate. For a competitive assay that same zero standard is B0, the maximum signal, and is not subtracted. In both formats the 4PL is fitted to the standards above zero, because zero has no place on a log axis; the zero standard is still listed in the standard results.