Analysis of the functional activity of hFIX expressed in macaques. Schematic of the functional assay, which relies on the ability of the polyclonal rhesus anti-hFIX antibodies to selectively capture hFIX in rhesus plasma, which is then activated by activated factor XI. This was incubated with factor X (FX) in the presence of cofactor (FVIIIa) and phospholipids to generate activated FX (FXa). The amount of FXa is quantitated using chromogenic substrate S2765. (B) A typical standard curve obtained with our functional immunocapture assay demonstrating a relatively linear range for detection of human FIX over 1% to 50% of normal levels using dilutions of human NPP in naive rhesus plasma. (C) Western blot after affinity purification of equivalent amounts of rhesus plasma; 10% SDS-PAGE of affinity-purified hFIX. Lane 1, fresh frozen plasma (FFP) as a positive control; lane 2, sample from monkey M2-sc that received vector into the portal circulation; lane 3, sample from monkey M5-sc that received vector into the systemic circulation; lanes 4 to 6, naive rhesus plasma from 3 animals that had not been transduced with AAV vectors; lane 7 represents 2 mU of affinity-purified hFIX (Replenine) as an additional positive control.