title: Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.
abstract: The ability to detect single protein molecules 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 immunocomplexes (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 approximately 10-20 enzyme-labeled complexes in 100 microl of sample (approximately 10(-19) M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10(-15) M) much lower than conventional ELISA. 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).
authors.affiliation: Quanterix Corporation, Cambridge, Massachusetts, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Rissin
authors.forename: Cheuk W
authors.initials: CW
authors.lastname: Kan
authors.forename: Todd G
authors.initials: TG
authors.lastname: Campbell
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Howes
authors.forename: David R
authors.initials: DR
authors.lastname: Fournier
authors.forename: Linan
authors.initials: L
authors.lastname: Song
authors.forename: Tomasz
authors.initials: T
authors.lastname: Piech
authors.forename: Purvish P
authors.initials: PP
authors.lastname: Patel
authors.forename: Lei
authors.initials: L
authors.lastname: Chang
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Rivnak
authors.forename: Evan P
authors.initials: EP
authors.lastname: Ferrell
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Randall
authors.forename: Gail K
authors.initials: GK
authors.lastname: Provuncher
authors.forename: David R
authors.initials: DR
authors.lastname: Walt
authors.forename: David C
authors.initials: DC
authors.lastname: Duffy
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008297:Male; D008832:Microchemistry / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000097:blood; D011468:Prostatectomy
chemical_list: D001798:Blood Proteins; D017430:Prostate-Specific Antigen