Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

TLDR

Convenient, wash‑free protein detection methods that remain robust in complex biological samples are highly demanded, yet developing such assays is challenging. The study introduces the Proximity Extension Assay (PEA), a nucleic acid proximity‑based antibody assay designed to detect proteins in plasma. PEA employs antibody‑DNA conjugates that, when both antibodies bind the target, generate a PCR amplicon via DNA polymerase, and a crowding matrix further enhances binding for low‑affinity antibodies. PEA achieves femtomolar sensitivity, high specificity, a 5‑log dynamic range in 1 µL plasma, superior performance with 3′‑exonuclease polymerases, and multiplex validation.

Abstract

Convenient and well-performing protein detection methods for a wide range of targets are in great demand for biomedical research and future diagnostics. Assays without the need for washing steps while still unaffected when analyzing complex biological samples are difficult to develop. Herein, we report a well-characterized nucleic acid proximity-based assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples. Target-specific antibody pairs are linked to DNA strands that upon simultaneous binding to the target analyte create a real-time PCR amplicon in a proximity-dependent manner enabled by the action of a DNA polymerase. 3'Exonuclease-capable polymerases were found to be clearly superior in sensitivity over non-3'exonuclease ones. A PEA was set up for IL-8 and GDNF in a user-friendly, homogenous assay displaying femtomolar detection sensitivity, good recovery in human plasma, high specificity and up to 5-log dynamic range in 1 μL samples. Furthermore, we have illustrated the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-affinity antibodies. Assay performance was also confirmed for a multiplex version of PEA.