BTN4406-BM-Mora R2.indd

Protein expression monitoring can be a good tool to determine disease onset and progression, and is also used in drug discovery and investigational science (1–4). However, commonly used assays lack the sensitivity and specificity required for the detection of low-abundance proteins (5,6). Several signal amplification technologies have been designed and applied to protein detection assays, including rolling-circle amplification (RCA) (6,7), tyramide signal amplification (TSA) (8), and enzyme-linked immunosorbent assay (ELISA) coupled with PCR (9). Although all technologies have successfully provided signal amplification to protein detection assays, none has been widely applied to microtiter plate or microarray ELISAs, presumably due to the required additional steps and/or equipment compared with the standard assay. 3DNA dendrimers are highly branched structures, constructed from seven different single strands of DNA. Some strands are complementary to each other in the middle section, and when incubated under the right conditions they hybridize, forming “X”-like structures called monomers. Monomers are the building blocks of the dendrimer. Different monomers hybridize through their single-stranded regions to form the layers of the dendrimer (10). The 3DNA dendrimer technology from Genisphere (Hatfield, PA, USA) has been established as a robust system to provide signal amplification for RNA detection (11–13). Here, we present an application of the 3DNA dendrimer technology to protein detection. Oligonucleotide-conjugated antibodies (for selectivity) and enzymes or dyes (for signal) are hybridized to the outer arms of the dendrimer, which functions as a core. Figures 1, A and B, show a schematic representation of the end product, called UltraAmp (Genisphere), and its assembly. UltraAmp reagents are prepared by combining a stock solution of the dendrimer with solutions of the antibody and enzyme-conjugated oligonucleotides. The buffer is spiked with NaCl to a final concentration of 200 mM to enhance the stability of the UltraAmp complex. After 20 min incubation, the solution is diluted to 6 ng/μL as dendrimer with PBS and 50% v/v final SuperFreeze (Pierce Biotechnology, Inc. Rockford, IL, USA). Standard sandwich ELISAs were run as recommended by the manufacturer (R&D Systems, Minneapolis, MN, USA). In UltraAmp assays, all standard conditions were maintained, except for the incubation with streptavidin horseradish peroxidase (SA-HRP; R&D Systems), which was replaced with UltraAmp Anti-biotin HRP (Cat. no. AB2080; Genisphere) diluted in Binding Buffer I (Cat. no. ABB100; Genisphere) to a final dendrimer concentration of 0.6 ng/μL. Each well was incubated with 50 μL of diluted UltraAmp reagent for 1 h on a reciprocating shaker at approximately 100 rpm at room temperature (RT). The wells were emptied, washed 5–6 Protein detection enhanced by 3DNA dendrimer signal amplification