血浆中成纤维细胞活化蛋白α和脯氨酰寡肽酶联合动力学荧光活性测定的开发和验证。,Clinica Chimica Acta

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血浆中成纤维细胞活化蛋白α和脯氨酰寡肽酶联合动力学荧光活性测定的开发和验证。
Clinica Chimica Acta ( IF 3.2 ) Pub Date : 2019-04-15 , DOI: 10.1016/j.cca.2019.04.063
An Bracke ₁ , Roos Van Elzen ₁ , Pieter Van Der Veken ₂ , Koen Augustyns ₂ , Ingrid De Meester ₁ , Anne-Marie Lambeir ₁

Affiliation

背景技术成纤维细胞活化蛋白α（FAP）被认为是各种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，已在（神经）炎症，神经变性以及癌症中进行了研究。当前的测定法不能充分地区分FAP和PREP，因此需要一种能够可靠地定量血浆中FAP / PREP活性比的测定法。方法在特征明确的PREP和FAP抑制剂存在下，在人EDTA血浆中测量FAP和PREP活性。结果在以Z-Gly-Pro-AMC为底物的条件下，开发了一种组合动力学测定法，以最佳地测量FAP和PREP活性。FAP-PREP组合检测的检出限为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分析进行比较来证明准确性。溶血以1.5 g / L血红蛋白作为临界值干扰了测定。在样品制备过程中，血小板和红细胞裂解后，PREP（而非FAP）活性会增加。结论采用这种新的测定方法，血浆中平均67％的Z-Gly-Pro-AMC转化活性可归因于FAP。

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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.

更新日期：2019-11-01

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