Improved immunoassay sensitivity and specificity using single-molecule colocalization

Amani A. Hariri, Sharon S. Newman, Steven Tan, Dan Mamerow, Alexandra M. Adams, Nicolò Maganzini, Brian L. Zhong, Michael Eisenstein, Alexander R. Dunn () and H. Tom Soh ()
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Amani A. Hariri: Stanford University
Sharon S. Newman: Stanford University
Steven Tan: Stanford University
Dan Mamerow: Stanford University
Alexandra M. Adams: Stanford University
Nicolò Maganzini: Stanford University
Brian L. Zhong: Stanford University
Michael Eisenstein: Stanford University
Alexander R. Dunn: Stanford University
H. Tom Soh: Stanford University

Nature Communications, 2022, vol. 13, issue 1, 1-11

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-32796-x

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