Improvement of sensitivity and dynamic range in proximity ligation assays by asymmetric connector hybridization.

The proximity ligation assay (PLA) is one of the most sensitive and simple protein assays developed to date, yet a major limitation is the relatively narrow dynamic range compared to other assays such as enzyme-linked immunosorbent assays. In this work, the dynamic range of PLA was improved by 2 orders of magnitude and the sensitivity was improved by a factor of 1.57. To accomplish this, asymmetric DNA hybridization was used to reduce the probability of target-independent, background ligation. An experimental model of the aptamer-target-connector complex (apt(A)-T-apt(B)-C(20,PLA)) in PLA was developed to study the effects of asymmetry in aptamer-connector hybridization. Connector base pairing was varied from the PLA standard of 20 total bases (C(20)) to an asymmetric combination with 15 total bases (C(15)). The results of this model suggested that weakening the affinity of one side of the connector to one aptamer would significantly reduce target-independent ligation (background) without greatly affecting target-dependent ligation (signal). These predictions were confirmed using PLA with asymmetric connectors for detection of human thrombin. This novel, asymmetric PLA approach should impact any previously developed PLA method (using aptamers or antibodies) by reducing target-independent ligation events, thus generally improving the sensitivity and dynamic range of the assay.