Improved immunoassay sensitivity and specificity using single-molecule colocalization.

Autor: Hariri AA; Department of Radiology, Stanford University, Stanford, CA, 94305, USA., Newman SS; Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.; Department of Electrical Engineering, Stanford University, Stanford, CA, 94305, USA., Tan S; Department of Chemical Engineering, Stanford University, Stanford, CA, 94305, USA., Mamerow D; Department of Radiology, Stanford University, Stanford, CA, 94305, USA., Adams AM; Department of Chemical Engineering, Stanford University, Stanford, CA, 94305, USA., Maganzini N; Department of Electrical Engineering, Stanford University, Stanford, CA, 94305, USA., Zhong BL; Department of Chemical Engineering, Stanford University, Stanford, CA, 94305, USA., Eisenstein M; Department of Radiology, Stanford University, Stanford, CA, 94305, USA.; Department of Electrical Engineering, Stanford University, Stanford, CA, 94305, USA., Dunn AR; Department of Chemical Engineering, Stanford University, Stanford, CA, 94305, USA. alex.dunn@stanford.edu., Soh HT; Department of Radiology, Stanford University, Stanford, CA, 94305, USA. tsoh@stanford.edu.; Department of Electrical Engineering, Stanford University, Stanford, CA, 94305, USA. tsoh@stanford.edu.; Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. tsoh@stanford.edu.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2022 Sep 12; Vol. 13 (1), pp. 5359. Date of Electronic Publication: 2022 Sep 12.
DOI: 10.1038/s41467-022-32796-x
Abstrakt: Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.
(© 2022. The Author(s).)
Databáze: MEDLINE
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