Multiplexed Immunoassays

Immunoassay has been widely used for quantification of proteins and small molecules in medical diagnostics, proteomics, drug discovery, and various biological research.1 ELISA (Enzyme-Linked ImmunoSorbant Assay) is the most commonly used immunoassay format.2 Traditional ELISA assays used in clinical settings are laborious and expensive and often consume large quantities of reagents and patient specimen. Meanwhile, armed with the knowledge from genomic and proteomic study, there is an increasing demand for technologies that are capable of extracting high density of bio-information from limited sample volume for better disease diagnosis, prognosis and treatment.3 This demand has driven the development of low-cost, flexible and high throughput methods for simultaneous detection of multiple proteins in parallel in a single assay (multiplexed immunoassay). Although the first concept towards multiplexed immunoassay was already described in 1961 by Feinberg for “microspot’ test of antibody-antigen reaction in the thin agar films4, it was not demonstrated until 1989 when Ekins described microarray technology principles in the ‘ambient analyte theory’ and envisioned the immense potential application in biomedical research and clinical diagnostics5. Since then, a great number of multiplexing platforms have been developed. Particularly, recent advances in technology (e.g. fluidics, optics, automated sample handling device) and informatics have enabled a real high-throughput multiplexed immunoassay.6-8 Today, multiplexed immunoassays are becoming popular and have been widely used in basic biomedical research due to their advantages in performing a large number of different assays all in a single reaction vessel from a relatively smaller sample volume with high efficiency. Multiplexed immunoassays are also becoming important for clinical diagnostic purpose by identifying multiple biomarkers for a wide range of diseases. Patterns of several biomarkers have better predictive value compared to detection of single analyte using ELISAs. Although there are still some challenges (e.g. complexity, expensive, validation requirement) for these tests, multiplexed protein test panels are now slowly penetrating into clinical diagnostics market and the time of their significant implementation is probably about to come. Several commercial multiplexed immunoassay platforms are available on this emerging market, including Luminex bead based platform, Meso Scale Discovery’s Multi Array Technology, and protein array platforms from Whatman, Arrylt and others.