Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium.
Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium.
Clin Chim Acta. 2019 Aug;495:154-160. doi: 10.1016/j.cca.2019.04.063. Epub 2019 Apr 11.
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.
FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors.
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.
With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.
成纤维细胞激活蛋白α（FAP）被认为是多种类型癌症的诊断和预后生物标志物。FAP 与脯氨酰寡肽酶（PREP）具有底物特异性，后者在（神经）炎症和神经退行性变以及癌症中均有研究。目前的检测方法不能充分区分 FAP 和 PREP，因此需要一种能够可靠地定量血浆中 FAP/PREP 活性比的检测方法。
在存在经过充分表征的 PREP 和 FAP 抑制剂的情况下，用人 EDTA 血浆中测量 FAP 和 PREP 活性。
开发了一种组合动力学测定法，在最佳条件下使用 Z-Gly-Pro-AMC 作为底物来测量 FAP 和 PREP 活性。该联合 FAP-PREP 测定的检测限为 0.009 U/L，定量限为 0.027 U/L。对于 PREP 和 FAP，批内变异系数分别为 3%和 4%，批间精密度分别为 7%和 12%。通过与已建立的终点测定法进行比较，证明了准确性。溶血会干扰测定，血红蛋白为 1.5 g/L 时作为截断值。在样品制备过程中血小板和红细胞的裂解会导致 PREP（而非 FAP）活性增加。
使用该新测定法，平均有 67%的 Z-Gly-Pro-AMC 转化活性可归因于 FAP。
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