Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations
Open accessAbstract
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).
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The presence of a given protein in a sample is inferred from identification of the peptides it contains, in a process known as protein inference.
“The presence of a given protein in the sample is inferred from identification of the peptides it contains, in a process known as 'protein inference' 32 .”
From How many human proteoforms are there? (2018) · cites this paper as [32]
The paper’s assay description (section labeled Abstract; page unavailable) describes detecting proteins with antibody capture and enzyme-labeled complexes, not identifying peptides to infer which proteins are present. Searches for “protein inference,” peptide identification, and mass spectrometry found no passage addressing the claimed process. (LLM, conf=0.75)
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- Authors
- Rissin, D. M., Kan, C. W., Campbell, T. G., Howes, S. C., Fournier, D. R., Song, L., Piech, T., Patel, P. P., Chang, L., Rivnak, A. J., Ferrell, E. P., Randall, J. D., Provuncher, G. K., Walt, D. R., & Duffy, D. C.
- Affiliations
- Quanterix (United States) (US)
- Tufts University (US)
- Venue
- Nature Biotechnology
- Published
- 2010-05-23
- Volume
- 28
- Issue
- 6
- Pages
- 595-599
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC2919230
- OpenAlex
- W2092198587
- Cited by
- 2,119
- References
- 25
- Citations by year
- 2026: 176 · 2025: 217 · 2024: 177 · 2023: 189 · 2022: 186 · 2021: 191 · 2020: 200 · 2019: 140 · 2018: 130 · 2017: 112 · 2016: 90 · 2015: 68 · 2014: 72 · 2013: 64 · 2012: 51
- FWCI
- 22.16
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (R43CA133987, R44CA133987)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Keywords
- single-molecule detection, digital ELISA, single-molecule fluorescence microscopy, antibody-coated beads, prostate-specific antigen, prostate cancer biomarkers, bead-based immunoassay
- MeSH terms
- Blood Proteins, Enzyme-Linked Immunosorbent Assay, Humans, Male, Microchemistry, Prostatectomy, Prostate-Specific Antigen
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