Enzymatic Change for Quantifying Analytes
There are three different formats in common use as detection systems in ELISAs: colorimetric assay, luminescence and fluorescence. As described in this Corning application note, Selecting the Detection System – Colorimetric, Fluorescent, Luminescent Methods for ELISA Assay, all rely on a method that shows a quantifiable change in the reaction medium, usually chromogenic, which is dependent on the concentration of the analyte. Developing the color change takes place at the end of the ELISA process with the addition of an enzyme substrate reagent that reacts with the enzyme-linked reporter.
ELISA Reporter Molecules
The most common ELISA reporters conjugated with the antibody are horse radish peroxidase (HRP), alkaline phosphatase (AP), and beta-D-galactosidase (BG). All three are stable, inert under standard assay conditions, and relatively easy to use.
HRP is a small molecule, and as such, when it's conjugated with the assay antibody, it does not cause steric hindrance and interfere with antigen binding. It is, however, affected by certain common preservatives, such as sodium azide, and can also be influenced by endogenous peroxidase activity in biological samples. Ensuring an adequate wash step during the ELISA usually mitigates interference.
AP is a larger molecule, so care must be taken to avoid steric hindrance. Detection also requires stricter conditions, so AP-specific assay buffers should be used. BG is an even larger molecule but is preferred for hydrophobic membrane surfaces such as dot blots, since the alcohol used as a wetting agent enhances detection.
Laboratory ELISA Detection Methods
In the lab, ELISA assays are read by measuring the action of the reporter molecule on an enzyme substrate added as the final step in the assay. The choice of substrate depends on the assay format. For example, an insoluble end result is best for membrane-bound reactions such as dot blots.
Other considerations include the range in expected concentrations, assay timing, sensitivity, and detection device in use. For example, with a wide range of concentrations expected, using an enzyme substrate that reacts over 15 to 30 minutes may give the best results.
Sensitive assays with very low analyte concentrations require fast-acting substrates. Assays with a timed endpoint include a termination step that stops and stabilizes the reaction prior to reading. It's also possible to monitor the rate of conversion by taking multiple measurements over a timed period to show kinetic analysis.
Colorimetric assay detection shows a color change that can be quantitated in the visible light spectrum, where optical density varies with concentration. It is important to incubate consistently to allow all wells to develop equally before reading the plate.
Fluorescence immunoassays use enzyme substrates that fluoresce when excited by certain light wavelengths. They are usually as sensitive as colorimetric assays but are not limited by higher analyte concentrations. In other words, they can glow more strongly without overwhelming signal detection, thus giving an accurate reading.
Luminescence detection in ELISAs relies on bioluminescence, where the light is created by biological substrates, or by chemiluminescence where the photons are released following a chemical reaction. Luminescence is considered the most sensitive method for ELISA detection due to the technique's compatibility with signal multiplication and amplification.
