Details

Autor(en) / Beteiligte

• Duffy, David C
• Rissin, David M
• Kan, Cheuk W
• Campbell, Todd G
• Howes, Stuart C
• Fournier, David R
• Song, Linan
• Piech, Tomasz
• Patel, Purvish P
• Chang, Lei
• Rivnak, Andrew J
• Ferrell, Evan P
• Randall, Jeffrey D
• Provuncher, Gail K
• Walt, David R

Titel

Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

Ist Teil von

• Nature biotechnology, 2010-06, Vol.28 (6), p.595-599

Ort / Verlag

New York, NY: Nature Publishing Group

Erscheinungsjahr

2010

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 ∼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 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).