Selective Measurement of Human Factor IX Padua Activity in Citrated Goat Plasma

Objectives: Nonclinical studies supporting a potential gene therapy treatment of Hemophilia B required the measurement of human factor IX (FIX) Padua in citrated goat plasma. Recently, we have described an anti-human FIX Padua Fab fragment, which was shown to selectively bind human FIX Padua even in the presence of excess human, macaque monkey and mouse wild-type FIX. Here, we present data on the measurement of human FIX Padua in citrated goat plasma.

Methods: Plate-adsorbed anti-FIX Padua-specific Fab fragment was used to capture human FIX Padua. Binding of FIX Padua was then quantified by a biotinylated anti-human FIX antibody and streptavidin peroxidase (FIX Padua ELISA) or by a chromogenic FIX activity test (FIX Padua activity test). Purified recombinant human FIX Padua, serially diluted in buffer, was used in both cases to establish assay calibration. Spike-recovery of FIX Padua was carried out in 10% citrated goat plasma to evaluate the assays.

Results: Both the FIX Padua ELISA and the FIX Padua activity test demonstrated adequate calibration curves combining required sensitivity (0.85 ng/mL and 0.21 mU/mL for the ELISA and the activity assay, respectively) and accuracy (back-fitted calibrators showed 100 ± 20% agreement to nominal concentrations). Citrated goat plasma, diluted 1/10, did not show any response in both systems. Interestingly, FIX Padua recoveries differed and were acceptable for the activity assay (recoveries ≥ 85%; n = 6; two FIX Padua concentrations). Furthermore, the dose-response curves obtained for the spiked samples showed good parallelism to the calibration curve. In contrast, FIX Padua recovery reached only 49% for the ELISA.

Conclusions: The Fab fragment -mediated capture of human FIX Padua followed by the chromogenic activity assay provided selective and accurate measurement of FIX Padua activity in citrated goat plasma, while the ELISA approach did not provide acceptable recovery of FIX Padua

No conflict of interest has been declared by the author(s).