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The standard curve is prepared by making serial dilutions of one known concentration of the analyte across a range of concentrations near the expected unknown concentration. The concentration of unknown samples is determined by interpolation which relies on a properly generated standard curve.

Polystyrene or polyvinyl chloride 96-well flat-bottomed plates are most commonly used for ELISAs. Using the right kind of ELISA plate helps maintain consistency, minimizes both edge and hook effects and maintains conditions required for downstream optical data collection steps.

An ELISA plate map is essentially a grid with 8 rows going down (A – H) and 12 rows going across (1 – 12). Each well can be identified by its corresponding letter and number such as B10. This well would be in the second row from the top and 10 columns from the left.

To obtain the ELISA standard curve, the absorbance (log10) is plotted on the y-axis against the concentration (log10) on the x-axis. Within the standard range, you can read off the concentration of your sample as indicated by the arrow. In this example, an OD of 0.5 gives a concentration of around 45 pg/ml.

After Running the ELISA A standard curve (aka calibration curve) for the protein of interest is constructed by plotting the mean absorbance (y-axis) against the protein concentration (x-axis) and choosing the best fit curve for the data points.

A microplate with a minimum protein-binding capacity of 400 ng/cm² and CV value (coefficient of variation) <5% is preferred. The choice of plate color depends upon the signal being detected. Plates should be inspected visually before use.

There are several formats used for ELISAs. These fall into either direct, indirect, or sandwich capture and detection methods. The key step is immobilization of the antigen of interest, accomplished by either direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate.

When you analyze ELISA data with quantitative ELISA kit, you have to plot the mean absorbance against the protein concentration and draw a curve fitting your standard result best, and then interpolate absorbance of samples to the curve to calculate the concentration.

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum.