Detection of IgG using AlphaLISA, an alternative to ELISAs
ELISAs work by having a primary antibody recognize its antigen and then a secondary antibody recognizes the primary antibody. The secondary antibody has an attached tag that is used to measure its presence. This linked tag is usually an enzyme that uses a colorimetric substrate that can be detected with a spectrometer (application note 184), or it can be a fluorescent tag that can be detected with a fluorometer (application note 213).
A newer ELISA format uses a third type of tag that is based upon AlphaScreen® technology from Perkin Elmer and it is known as AlphaLISA®. This type of ELISA has a more stable signal and the ratiometric aspect of the data analysis allows for a theoretical lower limit of detection compared to absorbance based ELISAs. Using the PHERAstar Plus HTS microplate reader and its dedicated AlphaScreen® Laser, different concentrations of an IgG antibody were detected using AlphaLISA®. Using 384 well plates, the Z’ value was >0.90 and the LOD was ~0.25 ng/mL of IgG antibody.
For more details on how to perform AlphaLISA® on the PHERAstar Plus, please see application note 190. For more general information on AlphaScreen and AlphaLISA® see our Assay Technologies page.