A single molecule assay for ultrasensitive detection of Fn14 in human serum.

Fibroblast growth factor inducible-14 (Fn14) is a receptor protein that plays an important role in the progression of cancer and some other diseases. Here, an ultrasensitive assay was developed for the detection of Fn14 based on a digital sandwich enzyme-linked immunosorbent bead-based assay (dELISA). Beads containing a single immunocomplex are loaded into microwells (~46 fL) and produce fluorescence through enzyme-catalyzed reactions in extremely small volumes. By measuring the number of fluorescent microwells in arrays arranged on a circular Disc, the concentration of Fn14 was determined. To obtain better performance for Fn14 detection, assay conditions including reagent concentrations and measurement parameters were optimized and 44 different antibody pairs were screened. The detection range of Fn14 is 1.26 pg/mL to 3683 pg/mL with a lower detection limit of 0.32 pg/mL, which is much lower than that of conventional ELISAs. In addition, the total operation of this assay is automated and only takes approximately an hour to accomplish. Furthermore, this assay was successfully applied to the determination of spiked Fn14 in serum samples with satisfactory performance.