Improved immunoassay sensitivity and specificity using single-molecule colocalization

• Published in: Nature communications 2022-09, Vol.13 (1), p.5359-5359, Article 5359
• Main Authors: Hariri, Amani A., Newman, Sharon S., Tan, Steven, Mamerow, Dan, Adams, Alexandra M., Maganzini, Nicolò, Zhong, Brian L., Eisenstein, Michael, Dunn, Alexander R., Soh, H. Tom
• Format: Article
• Language: English
• Subjects: 14/34; 49/1; 631/1647/2196; 631/1647/245/2225; 631/1647/328/2239; 631/1647/664/1467; 631/61/32; Antibodies; Binding; Enzyme-Linked Immunosorbent Assay - methods; Enzymes; Fluorescence; Fluorescence microscopy; Humanities and Social Sciences; Immunoassay; Immunoassay - methods; Immunoassays; multidisciplinary; Proteins; Science; Science (multidisciplinary); Sensitivity and Specificity; Tumor necrosis factor-α
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-32796-x

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 Si ngle- M olecule C olocalization A ssay (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. A major challenge of enzyme-linked immunosorbent assays is discriminating true signal from non-specific binding. Here the authors present a Single-Molecule Colocalization Assay (SiMCA) which eliminates such effects, enabling reproducible detection of picomolar protein concentrations.