ELISA Data Analyzer

4-Parameter Logistic (4PL) Curve Fitting

Enter your standard curve data, fit a 4PL model, and back-calculate unknown sample concentrations. Select an assay preset or enter custom standards.

Standard Curve Data

Enter concentration and OD values. For replicates, enter comma-separated OD values (e.g. 1.85, 1.92, 1.88).

# Concentration (pg/mL) OD Values (comma-separated replicates) Mean OD %CV
Frequently Asked Questions
A 4-parameter logistic (4PL) curve is a sigmoidal mathematical model used to fit dose-response data such as ELISA standard curves. The four parameters are: A (minimum asymptote, the response at zero concentration), B (Hill slope, describing the steepness of the curve), C (EC50/IC50, the concentration at the inflection point), and D (maximum asymptote, the response at infinite concentration). The equation is y = D + (A - D) / (1 + (x/C)^B). The 4PL model is preferred over linear regression for ELISA data because immunoassay dose-response curves are inherently sigmoidal, and linear fits only work over a narrow range.
A good ELISA standard curve should have an R-squared value above 0.99, ideally above 0.995. Each standard point should back-calculate to within 80-120% of its nominal concentration. The %CV between replicate wells should be below 20% for each standard. The curve should show a clear sigmoidal shape with well-defined upper and lower asymptotes. If more than one standard point fails acceptance criteria, the curve may need to be re-run.
LLOQ (Lower Limit of Quantification) is the lowest concentration of analyte that can be reliably measured with acceptable precision and accuracy. ULOQ (Upper Limit of Quantification) is the highest concentration that can be reliably measured. Together they define the quantifiable range or dynamic range of the assay. Samples with OD values outside this range should be flagged and re-assayed at a different dilution.
Samples are flagged when their OD values fall below the LLOQ or above the ULOQ of the standard curve. Below LLOQ means the analyte concentration is too low to quantify accurately. Above ULOQ means the response has saturated. To resolve this, dilute high-concentration samples further and re-assay, or use a more sensitive assay for low-abundance samples. Always report the dilution factor used.
For most ELISA applications, a 4PL fit is sufficient and recommended. The 5PL model adds an asymmetry parameter that can improve the fit when the sigmoidal curve is not symmetrical around the EC50. However, 5PL requires more data points and can overfit noisy data. Use 4PL as your default, and only switch to 5PL if you consistently see systematic deviations at the extremes of your curve with 7+ standard points.
A minimum of duplicate wells per standard concentration is standard practice, with triplicates recommended for critical assays or assay validation. Duplicates allow %CV calculation but provide limited statistical power. Triplicates give better precision estimates and allow outlier identification. For GLP/GMP-regulated assays, duplicates are the minimum requirement. The blank (zero standard) should always be run at least in duplicate.

About This ELISA Data Analyzer

This free ELISA data analysis tool uses a 4-parameter logistic (4PL) regression model to fit your standard curve and back-calculate unknown sample concentrations. The 4PL model is the gold standard for immunoassay data analysis, recommended by regulatory agencies including the FDA and EMA for bioanalytical method validation.

The tool supports common ELISA formats including sandwich ELISA, competitive ELISA, and direct/indirect detection. Pre-configured assay presets are available for popular analytes including human IgG, IL-6, TNF-alpha, CHO Host Cell Proteins (HCP), and residual Protein A — all commonly measured in biopharmaceutical process development and quality control.

Key features include automated 4PL curve fitting with Levenberg-Marquardt optimization, back-calculation accuracy validation, LLOQ/ULOQ determination, %CV flagging for replicate precision, and comprehensive export options. All calculations run entirely in your browser — no data is uploaded to any server.