Single-molecule Enzyme-linked Immunosorbent Assay Detects Serum Proteins At Subfemtomolar Concentrations
Nature Biotechnology | May 23, 2010
David M Rissin, Cheuk W Kan, Todd G Campbell, Stuart C Howes, David R Fournier, Linan Song, Tomasz Piech, Purvish P Patel, Lei Chang, Andrew J Rivnak, Evan P Ferrell, Jeffrey D Randall, Gail K Provuncher, David R Walt & David C Duffy
The ability to detect single protein molecules1, 2 in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immune complexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as ~10–20 enzyme-labeled complexes in 100 µl of sample (~10-19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10-15 M) much lower than conventional ELISA3, 4, 5. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).