Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.
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).
Rissin
David M
DM
Quanterix Corporation, Cambridge, Massachusetts, USA.
Kan
Cheuk W
CW
Campbell
Todd G
TG
Howes
Stuart C
SC
Fournier
David R
DR
Song
Linan
L
Piech
Tomasz
T
Patel
Purvish P
PP
Chang
Lei
L
Rivnak
Andrew J
AJ
Ferrell
Evan P
EP
Randall
Jeffrey D
JD
Provuncher
Gail K
GK
Walt
David R
DR
Duffy
David C
DC
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
D001798:Blood Proteins; D017430:Prostate-Specific Antigen