ELISA Calculator

ELISA (Enzyme-Linked Immunosorbent Assay) is a lab technique used to detect and quantify proteins, antibodies, or antigens in a sample — and this calculator processes your raw plate reader data into a calibration curve and sample concentrations. Paste your Raw OD Data from your plate reader, then configure your Standards, dilution factor, replicate settings, and curve fitting method (Linear, Polynomial, 4PL, or 5PL). The ELISA Calculator returns your R² goodness-of-fit, the curve equation, detection limit, and coefficient of variation.

Enter raw optical density values from your ELISA plate reader

ng/ml

Results

R² (Coefficient of Determination)

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Curve Equation

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Detection Limit

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Coefficient of Variation

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Results Table

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Frequently Asked Questions

What is 4PL curve fitting and when should I use it?

Four Parameter Logistic (4PL) is the most common curve fitting method for ELISA assays. It produces an S-shaped curve that better fits the typical dose-response relationship in immunoassays, especially when dealing with high and low concentration ranges.

How do I interpret the R² value in my ELISA results?

R² (coefficient of determination) indicates how well your standard curve fits the data points. Values closer to 1.0 indicate better fit. Generally, R² values above 0.95 are considered acceptable for ELISA assays, with values above 0.99 being excellent.

What format should I use for entering my OD data?

Enter your optical density values as they appear from your plate reader, typically in a 12x8 matrix format. Separate each value with a space or tab. You can copy and paste directly from Excel or Word documents.

How many replicates should I use for standards and samples?

For standards, duplicate or triplicate measurements are recommended to ensure accuracy of the standard curve. For samples, duplicates are typically sufficient, though triplicates provide better precision for critical measurements.

What is the coefficient of variation (CV) and what's acceptable?

CV measures the variability between replicate measurements. For ELISA assays, CV values below 10% are generally acceptable for standards, while CV values below 15% are acceptable for samples.

How do I choose the right dilution factor for my standards?

The dilution factor depends on your analyte's concentration range. 1:2 serial dilutions provide more data points and better curve resolution, while 1:10 dilutions cover broader concentration ranges with fewer standards.

What should I do if my standard curve has poor fit?

Poor curve fit can result from pipetting errors, expired reagents, or inappropriate dilution ranges. Check your standard preparation, ensure proper mixing, and consider using different curve fitting methods or adjusting your concentration range.

Can I use this calculator for other immunoassay types besides ELISA?

Yes, this calculator works for any assay that uses a standard curve approach, including chemiluminescent immunoassays, fluorescent assays, and other plate-based immunoassays that measure dose-response relationships.