When measuring ELISAs with a microplate reader, usually just one specific absorbance wavelength per well is read. This reading is then compared to a standard curve to determine the relative concentration of each well, or it is compared to a blank or negative control to show a change from a baseline response. Since different ELISA enzymes have different substrates (see yesterday’s blog) the specific wavelength that is measured will be different. In addition, depending on the assay conditions, the specific recommended wavelength may not correspond to the absorbance maximum wavelength.
With full spectrum analysis, selecting the exact or optimal wavelength is no longer a concern because all wavelengths can be captured at once in less than one second per well. Using full spectrum analysis, researchers can choose the best wavelength after the ELISA data is collected. Then all manners of data calculations can be done to improve assay performance.
This application note details how full spectrum analysis benefits ELISAs as well other classic absorbance assays: Old Assays, New Instrument: ELISA; NADH and NADPH Conversion; DNA and Protein Quantitation.