RT Journal Article
SR Electronic
T1 Improved immunoassay sensitivity and specificity using single-molecule colocalization
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.12.24.474141
DO 10.1101/2021.12.24.474141
A1 Amani A. Hariri
A1 Sharon S. Newman
A1 Steven Tan
A1 Dan Mamerow
A1 Michael Eisenstein
A1 Alexander Dunn
A1 H. Tom Soh
YR 2021
UL http://biorxiv.org/content/early/2021/12/25/2021.12.24.474141.abstract
AB Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still suffers some notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background arising from the interaction of detection antibodies with the assay substrate or interferents in the sample matrix. Single-Molecule Colocalization Assay (SiMCA) overcomes this problem by employing total internal reflection fluorescence (TIRF) microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized fluorescent signals, we can essentially eliminate the confounding effects of background produced by non-specific binding of detection antibodies. We further employed a normalization strategy to account for the heterogeneous distribution of the capture antibodies, greatly improving the reproducibility of our measurements. In a series of experiments with TNF-α, we show that SiMCA can achieve a three-fold lower limit of detection compared to conventional single-color assays using the same antibodies and exhibits consistent performance for assays performed in complex specimens such as chicken serum and human 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.Competing Interest StatementThe authors have declared no competing interest.