The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma

Background: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro) inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma. Methods: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors. Results: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation. Conclusion: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.