Detection of hFIX in rhesus plasma.

(A) Schematic representation of ELISA in which hFIX in rhesus plasma was captured by anti-hFIX Abs (capture Ab). The level of hFIX antigen was then determined with an HRP-conjugated goat anti-hFIX polyclonal secondary Ab. (B) A typical standard curve obtained with our ELISA showing a relatively linear range for detection of hFIX above 1% to 25% of normal levels. (C) Western blot analysis. Rhesus plasma was spiked with a known concentration of hFIX (0-3000 ng/mL). The vitamin K–dependent proteins were then precipitated by using barium citrate, and 5 μg precipitated protein was analyzed by a Western blotting procedure using rhesus immune serum (RQ1305) as the primary Ab. A band (∼70 kDa) in the expected position for FIX on reducing gels was detected only in rhesus plasma spiked with hFIX.