Calculating & Analyzing ELISA data
Calculating & analysing ELISA data
Turn raw optical-density readings into reliable, publishable numbers. Learn how to build a standard curve, read concentrations, and validate your data with %CV and spike recovery.
Key takeaways
ELISA reliability hinges on an accurate standard curve and a consistent protocol.
Positive and negative controls plus the coefficient of variation (%CV) confirm precision, while spike recovery tests sample-matrix compatibility.
Calculating results from ELISA data
Data are presented as a plot of optical density (OD) versus the log concentration of the sample. Standards of known concentration generate a standard curve from which unknown analyte concentrations are read.
Run samples in duplicate or triplicate for statistical validation, and always include controls. Negative controls contain no analyte; positive controls contain a known amount of analyte.
Preparing an ELISA standard curve
After running the protocol and reading your samples and standards on a plate reader, plot the standard curve: mean absorbance (Y axis) versus the known concentration of the standards (X axis). Use software to draw a best-fit curve.
For a sandwich ELISA, OD450 increases with the amount of analyte. For a competitive ELISA, OD450 decreases as analyte concentration rises.
Calculating the absorbance
Read unknown concentrations from your standard curve in seven steps.
Coefficient of variation (%CV)
The coefficient of variation flags inconsistency in your results, expressed as a percentage of the variance relative to the mean - the larger the value, the greater the error. It is the ratio of the standard deviation (σ) to the mean (µ): CV = σ / µ.
A high %CV can be caused by microbial or reagent contamination, pipetting inaccuracy, wells drying out (cover plates during incubations) or temperature variation (incubate at a stable temperature away from drafts). Every Assay Genie kit is characterised for sensitivity, range and inter/intra-assay %CV.
Spike recovery
Spike recovery tests whether components in your sample matrix interfere with antibody-antigen binding. The matrix is spiked with a known concentration of recombinant target, the ELISA is run, and the measured concentration is compared to expected.
Recovery is reported as a percentage; below ~80% generally indicates matrix interference and a different kit should be chosen. Assay Genie performs linearity and spike-recovery analysis on every ELISA kit batch.
ELISA kits & guides
Validated kits and further reading to support your analysis.
FAQs
How do I read an unknown concentration from the standard curve?
Find the sample's mean absorbance on the Y axis, draw a horizontal line to the best-fit standard curve, then drop a vertical line to the X axis to read the concentration. Multiply by any dilution factor used.
What is an acceptable %CV for an ELISA?
Replicates should generally be within 20% of the mean. A high %CV points to contamination, pipetting error, wells drying out or temperature variation.
What does spike recovery tell me?
It shows whether your sample matrix interferes with the assay. Recovery below about 80% suggests matrix interference and a different kit or further dilution may be needed.
Should I use a linear or 4PL/5PL curve fit?
Use the best-fit model for your data range; many ELISAs are non-linear, so a 4- or 5-parameter logistic (4PL/5PL) fit often describes the full curve better than a straight line.
Questions about your ELISA data?
Our PhD-level team can help with curve fitting, %CV and validating results for publication.