ELISA Calculator

How to Use the ELISA Calculator

1. Choose between Linear Fit (for simple, high-linearity assays) or 4PL Fit (for sigmoid ELISA curves — recommended for most sandwich ELISAs).
2. Enter at least 3 standard concentration/absorbance pairs. Click Load Example to see a sample dataset.
3. Select the concentration unit matching your standards (pg/mL, ng/mL, etc.).
4. Enter your sample absorbance values separated by commas.
5. Enter the dilution factor if samples were diluted before the assay.
6. Click Calculate ELISA Results to see the curve fit, R² value, and sample concentrations.

Linear vs 4PL Curve Fitting for ELISA

Most ELISA standard curves are sigmoid (S-shaped), especially at high and low concentrations where the signal saturates. The 4-parameter logistic (4PL) model accounts for this and provides a more accurate fit across the entire dynamic range.

The 4PL equation is: y = D + (A − D) / (1 + (x / C)^B), where A = minimum asymptote, B = Hill slope, C = EC50 (inflection point), D = maximum asymptote.

Linear fitting is appropriate only when you are working within the linear range of the assay (typically the middle portion of the sigmoid). If your standards span a wide concentration range, always use 4PL.

ELISA Data Quality Tips

• R² ≥ 0.99: A good standard curve should have R² ≥ 0.99 for linear fit, or strong convergence for 4PL.
• CV ≤ 15%: Run standards and samples in duplicate or triplicate and ensure the coefficient of variation (CV) is below 15%.
• Subtract blank: Always subtract the blank (zero standard) absorbance from all readings before entering data.
• Sample range: Samples with absorbance outside the range of your standards are flagged as out-of-range (OOR). Dilute them and re-run.
• Dilution factor: If you diluted your sample 1:10 before the assay, the actual concentration = calculated × 10.