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Improved immunoassay sensitivity and specificity using single-molecule colocalization.

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.

SUBMITTER: Hariri AA

PROVIDER: S-EPMC9468026 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Improved immunoassay sensitivity and specificity using single-molecule colocalization.

Hariri Amani A AA Newman Sharon S SS Tan Steven S Mamerow Dan D Adams Alexandra M AM Maganzini Nicolò N Zhong Brian L BL Eisenstein Michael M Dunn Alexander R AR Soh H Tom HT

Nature communications 20220912 1

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 fr ...[more]

PMID: 36097164

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Project description:The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

Dataset Information

Improved immunoassay sensitivity and specificity using single-molecule colocalization.

Publications

Improved immunoassay sensitivity and specificity using single-molecule colocalization.

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