ELISA Calculators

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ELISA (enzyme-linked immunosorbent assay) is a plate-based immunoassay that detects and quantifies proteins, hormones, antibodies, and small molecules in biological samples. It combines the specificity of antibody-antigen binding with enzyme-linked signal amplification, producing a colorimetric, fluorescent, or chemiluminescent readout proportional to analyte amount. ELISA is among the most widely used assays in clinical diagnostics, research, and drug development — applications range from HIV and pregnancy testing to cytokine profiling and pharmacokinetic drug monitoring.

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Four ELISA Formats

  • Direct: Antigen coated on plate; enzyme-conjugated detection antibody binds directly. Simple and fast; lower sensitivity; no secondary antibody needed.
  • Indirect: Antigen coated; unlabeled primary binds; enzyme-conjugated secondary detects the primary. Higher sensitivity via signal amplification.
  • Sandwich: Capture antibody on plate traps antigen; enzyme-linked detection antibody binds a second epitope. Highest sensitivity and specificity; most common format.
  • Competitive: Labeled antigen competes with sample antigen for antibody. Signal decreases as sample concentration increases — used when only one epitope is accessible.

ELISA Procedure

Coat plate → block (1–5% BSA or non-fat milk) → add sample → wash → add detection antibody → wash → add enzyme substrate → stop reaction → read OD. HRP (horseradish peroxidase) with TMB substrate is most common, read at 450 nm after H₂SO₄ stop.

Standard Curve and Quantification

Run serial dilutions of a purified standard alongside samples. Fit a 4-parameter logistic (4PL) curve to OD vs. concentration. Interpolate sample concentrations from the curve. Multiply by dilution factor. A well-optimized sandwich ELISA detects as low as 1–10 pg/mL.

Sensitivity Enhancement

Sensitivity can be improved using chemiluminescent or fluorescent substrates (10–100× over colorimetric TMB), biotin-streptavidin amplification, or digital ELISA platforms (Simoa) that detect fg/mL concentrations via single-molecule counting.

Glossary

ELISA
Enzyme-linked immunosorbent assay; a plate-based immunoassay using antibody-antigen binding and enzyme amplification to detect and quantify analytes in biological samples.
4-Parameter Logistic (4PL) Curve
A sigmoidal curve-fitting model used for ELISA standard curves; provides accurate concentration interpolation across the full dynamic range of the assay.
HRP (Horseradish Peroxidase)
The most common ELISA enzyme label; converts TMB substrate to a blue color (stopped to yellow with H₂SO₄); read at 450 nm; provides good sensitivity and reagent stability.

Frequently Asked Questions

A sandwich ELISA uses two antibodies recognizing different epitopes on the target: (1) Capture antibody coated on the plate surface binds the antigen from sample. (2) An enzyme-linked detection antibody (different epitope) binds the captured antigen. (3) Enzyme substrate is added and converted to a colored product. Absorbance is proportional to analyte concentration. Sandwich ELISA achieves pg/mL sensitivity and high specificity due to requiring two-antibody recognition.

Direct ELISA: enzyme is conjugated directly to the primary detection antibody — faster, fewer steps, but lower sensitivity. Indirect ELISA: an unconjugated primary antibody binds the antigen; then an enzyme-conjugated secondary antibody binds the primary. Multiple secondary antibodies can bind each primary, amplifying signal 2–10 fold. Indirect gives higher sensitivity but requires one more wash step and can have slightly more background.

Prepare 7–9 serial dilutions of a known-concentration standard spanning from near the detection limit to well above expected sample concentrations. Run in duplicate or triplicate on the same plate as samples. After running the ELISA, plot OD vs. concentration and fit a 4-parameter logistic (4PL) curve. Read sample concentrations by interpolation from the fitted curve, then multiply by any sample dilution factor applied before the assay.

Standard sandwich ELISA sensitivity is typically 1–100 pg/mL depending on antibody affinity and assay format. Sensitivity can be improved by using chemiluminescent substrates instead of colorimetric (10–100× gain), adding a biotin-streptavidin amplification step, or using single-molecule digital ELISA platforms (Simoa, Quanterix) that reach fg/mL detection. The lower limit of quantification (LLOQ) is defined as the lowest concentration giving a signal measurably above background with acceptable precision.