Immuno-PCR with digital readout

Stoichiometric constraints for detection of EV-borne biomarkers in blood

“…With the chemiluminescent ELISA, assays are reaching the sensitivity of 0.5 pg/mL in the lower quantification ranges [175], which would translate to 50 fg for a 100 µL reaction. Immuno-dPCR successfully detected target protein with a concentration of 0.4 pg/mL (~6 fg loaded in the reaction) [174]. Similar sensitivity was achieved with EV analyses [144], assuming that 38 EVs have total protein content of ~30 fg (according to our previous estimates), and that the targeted protein is representing just a fraction of that total.…”

“…So far, we can conclude that the bulk isolation and analysis of EVs from plasma with described technologies would be challenging but feasible, at least for some analyte molecules. The introduction of the signal amplification for protein molecules, by using DNA templated methodologies such as in immuno-PCR and immuno-sequencing, add unprecedented sensitivity to conventional immunoassays [174]. The Figure 2 and Table 5 shows the scale of analytical sensitivity of the state-of-art methodologies that are available for interrogation of EV molecular content, most of which are today used for bulk measurements.…”

Blood-based biomarkers for Alzheimer disease: mapping the road to the clinic

“…Since their concentration increases as tumor advances over time, they are useful dynamic indicators of tumor growth49. In proteomics, innovative methods of immuno-PCR, based on conjugations of specific antibodies and nucleic acids and exploiting ultrasensitive PCR signal amplification, can result into a 100 to 10,000-fold increase in sensitivity compared with the analogous enzyme-amplified immunoassays, hence considerably increasing the sensitivity of protein biomarker detection50,51. Powerful computational and integrative network biology tools are needed to assimilate the large multimodal information generated by the “omics” exploratory analyses and to comprehensively understand the decompensations at systems level52.…”

Digital detection of proteins

“…99 Byrnes et al 100 have also reported a digital droplet immunoassay based on PLA readout in polydisperse droplets, and used it to develop a wash-free assay for IL-8 with picomolar detection limits. Schröder et al 101 have digitized immuno-PCR using water-in-oil microdroplets, calling it Table 3 Summary of the approaches taken to solve the 3 main technical challenges in developing digital protein detection methods. Illustrative references are provided for each approach; the main text contains more comprehensive references…”

“…99 Byrnes et al 100 have also reported a digital droplet immunoassay based on PLA readout in polydisperse droplets, and used it to develop a wash-free assay for IL-8 with picomolar detection limits. Schröder et al 101 have digitized immuno-PCR using water-in-oil microdroplets, calling it digital droplet immuno-PCR (ddIPCR). They formed oligonucleotide-labeled immunocomplexes at the surface of an ELISA plate, chemically released the label from the surface, partitioned the labels into droplets using a commercial droplet generator, performed PCR on the droplets, and then analyzed the on/off status of the droplets using a commercial droplet reader.…”