2010 OAK RIDGE CONFERENCE, SAN JOSE, CA

Rissin DM1, Kan CW1, Campbell TG1, Howes SC1, Fournier DR1, Song L1, Piech T1, Patel PP1, Chang L1, Rivnak AJ1, Ferrell EP1, Randall JD1, Provuncher GK1, Walt DR2, Duffy DC1. 1Quanterix Corporation, Cambridge, MA, and 2Tufts University, Medford, MA.

We describe a method for detecting single immunocomplexes formed in the enzyme–linked immunosorbent assay (ELISA); we call this method digital ELISA. This method is based on isolating single immunocomplexes labeled with an enzyme in arrays of femtoliter wells, sealing the arrays in the presence of the enzyme substrate, and fluorescently imaging the array. Fluorescent product molecules of the enzyme–substrate reaction are confined in the femtoliter volume, giving rise to a local high concentration that can be easily detected using a standard fluorescent microscope. By using high density arrays of femtoliter wells, hundreds to thousands of single immunocomplexes can be detected simultaneously. Isolation of single immunocomplexes in this way gives rise to a dramatic increase in sensitivity to enzyme labels over bulk, ensemble detection methods. This method was used to detect yoctomole levels of ß–galactosidase enzyme label. The enzyme sensitivity using these single molecule arrays – which we term SiMoA – is ~105 times greater than the same enzyme measured on a fluorescent plate reader and over 100 times greater than detection of alkaline phosphatase using chemiluminescence, the gold–standard of sensitive ELISA detection. Ultra–sensitive immunoassays for detecting clinically–relevant proteins have been developed using digital ELISAs that can detect sub–femtomolar concentrations of the proteins in serum. These assays have been used to detect proteins in clinical samples at concentrations that are well below the detection limits of current immunoanalyzers. With the aim of detecting very low concentrations of nucleic acids without recourse to PCR (or other target amplification), a single molecule assay for DNA was also developed. Attomolar limits of detection for direct detection of single molecules of DNA in a sandwich assay were achieved. This single molecule detection technology couples directly to the back–end of established clinical immunoassays providing a two to four log improvement over the detection limit of current clinical methods. The method overcomes the complexity associated with existing ultra–sensitive protein detection methods, bringing single molecule sensitivity to clinical immunodiagnostics that should lead to downstream benefits in patient care.

Attomolar Detection of Proteins in Serum Using Single Molecule Enzyme–Linked Immunosorbent Assays