Which ELISA curve fit should I use?

For most sandwich and competitive ELISAs with a sigmoidal response, a 4-parameter logistic (4PL) fit is recommended. If your standards are clearly linear over the working range, you can use a linear or log-linear fit. Always inspect the standard curve and R² value to confirm the model is appropriate.

How are sample concentrations calculated from OD?

The calculator first fits a curve using the standard concentrations and their mean OD values. For each sample, it inverts that curve to find the concentration corresponding to the sample OD. If you specify a dilution factor, the tool multiplies the calculated concentration by that factor to give the concentration in the original sample.

What are common sources of error in ELISA data?

Common sources of error include pipetting inaccuracies, uneven plate washing, edge effects, incorrect incubation times or temperatures, expired or poorly stored reagents, and using OD readings outside the reliable range of the standard curve. Running standards and samples in replicates and checking CV% helps identify these issues.

ELISA Data Analysis Calculator – Standard Curve, Sample Concentration & QC

Q: What does this ELISA calculator do?

This ELISA calculator lets you enter standard concentrations and OD values, fits a standard curve (4PL, linear, or log-linear), and then calculates the concentration of your unknown samples, including dilution correction and basic quality-control metrics like R² and CV%.

Analyze ELISA plate data in your browser: build a standard curve, calculate sample concentrations, apply dilution factors, and review basic QC metrics.

How this ELISA calculator works

Enzyme-linked immunosorbent assay (ELISA) is a plate-based immunoassay used to quantify proteins, antibodies, hormones, and many other analytes. The core of ELISA data analysis is the standard curve, which relates optical density (OD) to known concentrations.

This tool helps you:

Enter standards and samples with multiple replicates
Fit a standard curve (4PL, linear, or log-linear)
Calculate sample concentrations from OD values
Apply dilution factors to report concentrations in the original sample
Review QC metrics such as R² and replicate CV%

Curve fitting models

Three models are implemented:

4-parameter logistic (4PL) – recommended for most sandwich and competitive ELISAs.
Linear – for assays that are linear over the working range.
Log-linear – linear fit of OD vs. log₁₀(concentration).

4-parameter logistic (4PL) equation

For concentration \(x\) and OD \(y\):
\[ y = D + \frac{A - D}{1 + \left(\frac{x}{C}\right)^B} \] where:

\(A\) = minimum asymptote (bottom OD)
\(D\) = maximum asymptote (top OD)
\(C\) = inflection point (EC\(_{50}\))
\(B\) = slope (Hill coefficient)

To calculate the concentration from an OD value, the equation is inverted:

\[ x = C \left( \left( \frac{A - D}{y - D} - 1 \right)^{1/B} \right) \]

Linear and log-linear models

For a linear model, the relationship is:

\[ y = m x + b \quad \Rightarrow \quad x = \frac{y - b}{m} \]

For a log-linear model, the fit is performed on log₁₀(concentration):

\[ y = m \log_{10}(x) + b \quad \Rightarrow \quad x = 10^{\frac{y - b}{m}} \]

Replicate statistics and CV%

For each standard and sample, the calculator computes the mean OD and coefficient of variation (CV%):

\[ \text{CV\%} = \frac{\text{standard deviation}}{\text{mean}} \times 100 \]

High CV% suggests pipetting or plate issues. Many labs aim for CV% < 10–15% for standards and < 15–20% for samples, but acceptable limits depend on the assay.

Step-by-step: analyzing an ELISA plate

Choose the curve model (4PL is usually best).
Set the number of standards and replicates, then click Apply layout.
Enter standard concentrations and OD values. Mark blanks if applicable.
Enter sample IDs, dilution factors, and OD values.
Click Fit curve & calculate concentrations.
Inspect the standard curve plot and R² value.
Review sample concentrations and flags (e.g., out-of-range OD).
Export the results as CSV for your records or LIMS.

Limitations and good practices

Always ensure OD values fall within the reliable range of the standard curve.
Repeat samples with very high or low OD (outside curve range) using an adjusted dilution.
Use consistent pipetting and plate handling to minimize variability.
Consult your kit’s instructions and lab SOPs for acceptance criteria and validation.

FAQ

What does this ELISA calculator do?

It fits a standard curve to your ELISA standards and uses that curve to convert sample OD values into concentrations, with optional dilution correction and QC metrics.

Which curve fit should I choose?

Use 4PL for most immunoassays with a sigmoidal response. Use linear or log-linear only if your standards clearly follow a straight line over the working range.

Can I use this for competitive ELISA?

Yes. Competitive ELISAs also typically follow a logistic relationship between OD and concentration. Just ensure you interpret the direction of the curve correctly (higher concentration often means lower OD).

Is this calculator a substitute for validated lab software?

No. This tool is for educational and research support only. For regulated assays or clinical decision-making, use validated, audited software and follow your institution’s quality system.

ELISA Data Analysis Calculator

1. Assay setup

2. Enter standards (concentration & OD)

3. Enter samples (OD & dilution)

4. Analyze

5. Results & QC

Standard curve

Sample concentrations