Spear principle

SPEAR (Successive Proximity Extension Amplification Reaction) is an ultrasensitive immunoassay platform that requires minimal sample volumes and can be implemented in any lab with qPCR capability.  

SPEAR utilizes two affinity probes that are conjugated to specially designed DNA sequences, SPEAR Probes A and B. When the two probes bind to their intended target, the end of Probe B hybridizes to its complementary region on Probe A. In the presence of a DNA polymerase, Probe B is extended until the polymerase encounters a chemical stopper sequence that terminates the extension. The extended Probe B then dissociates from Probe A, completing the first extension step. The newly extended region of Probe B then hybridizes to its complementary region on the other end of Probe A to repeat the extension process, completing the second extension step. This produces a double-extended Probe B that can be quantified through qPCR.  

To complete both extension steps, SPEAR requires the two affinity probes to stay in proximity long enough, about 1 to 2 minutes. Compared with conventional proximity assays, this reduces background due to random interactions of the two probes and delivers a sensitivity improvement of 2 to 3 orders of magnitude.