Proximity extension assay

antibodies and is a type of immunoassay. Target binding by the proximity probes increases their local relative effective concentration of the DNA-tags enabling hybridization of weak complementarity to each other which then enables a DNA polymerase mediated extension forming a united DNA sequence specific for each target protein detected. The use of 3'exonuclease proficient polymerases lowers background noise and hyper thermostable polymerases mediate a simple assay with a natural hot-start reaction. This created pool of extension products of DNA sequence forms amplicons amplified by PCR where each amplicon sequence corresponds to a target proteins identity and the amount reflects its quantity. Subsequently, these amplicons are detected and quantified by either real-time PCR or next generation DNA sequencing by DNA-tag counting. PEA enables the detection of many proteins simultaneously (so called multiplexing) due to the readout requiring the combination of two correctly bound antibodies per protein to generate a detectable DNA sequence from the extension reaction.^[5] Only cognate pairs of sequence are detected as true signal, enabling multiplexing beyond solid phase capture methods limited at around 30 proteins at a time.^[6] The DNA amplification power also enable minute sample volumes even below one microliter. PEA has been used in over 1000 research publications.^[7][8]