Roche Denis, Mesner Alexandra, Al Nakib Malik, Leonard Frederic, Beaune Philippe
AP-HP, Hôpital Européen Georges Pompidou, Service de Biochimie, 75015 Paris, France.
Clin Chem. 2009 Mar;55(3):513-8. doi: 10.1373/clinchem.2008.117002. Epub 2009 Jan 23.
Alpha1-Antitrypsin (A1AT) deficiency is currently detectable by protein immunoassay, phenotyping, and genotyping of the S and Z mutations, but no fully automated method for standard biochemical analyzers is yet available. Here, we present a method that measures the antitryptic activity in serum. This method is rapid, automated, and allows the easy evaluation of a large cohort of patients.
Our automated assay involves determining serum antitryptic capacity on the Olympus AU 400 autoanalyzer by using trypsin and succinylated gelatin as substrate in the presence of trinitrobenzene sulfonic acid. The results are expressed as a percentage of inhibition of the reaction of trypsin with succinylated gelatin. After we performed analytical validation studies and reference-interval determination based on serum samples from 120 healthy persons, we tested the assay on deidentified samples from 120 patients with various pathologies (primarily pulmonary) of unexplained origin and normal A1AT concentrations and phenotypes.
The analysis rate was up to 120 samples per hour. Intraassay CVs ranged from 3.1%-16.2%, and interassay CV was 7.5%. The reference population showed mean (SD) 58.4 (6.7)% inhibition. The detection limit was 9.5% inhibition. The 120 studied patients displayed significantly lower mean activity than 120 healthy individuals (P < 0.0001).
This assay is stable, reliable, and easily automated by use of open-system analyzers, allowing for the rapid evaluation of patients. After further validation on a larger randomized cohort, this new approach should function as a useful method to explore A1AT deficiency, especially in large-scale studies.
目前可通过蛋白质免疫测定、表型分析以及S和Z突变的基因分型来检测α1-抗胰蛋白酶（A1AT）缺乏症，但尚无适用于标准生化分析仪的全自动检测方法。在此，我们介绍一种可测定血清中抗胰蛋白酶活性的方法。该方法快速、自动化，且便于对大量患者进行评估。
我们的自动检测方法是在三硝基苯磺酸存在的情况下，以胰蛋白酶和琥珀酰化明胶为底物，在奥林巴斯AU 400自动分析仪上测定血清抗胰蛋白酶能力。结果以胰蛋白酶与琥珀酰化明胶反应的抑制百分比表示。在基于120名健康人的血清样本进行分析验证研究和参考区间测定后，我们对120例病因不明且A1AT浓度和表型正常的各种疾病（主要是肺部疾病）患者的匿名样本进行了检测。
分析速度可达每小时120个样本。批内变异系数范围为3.1% - 16.2%，批间变异系数为7.5%。参考人群的平均（标准差）抑制率为58.4（6.7）%。检测限为9.5%抑制率。120例研究患者的平均活性显著低于120名健康个体（P < 0.0001）。
该检测方法稳定、可靠，且可通过开放系统分析仪轻松实现自动化，能够对患者进行快速评估。在更大规模的随机队列中进一步验证后，这种新方法应成为探索A1AT缺乏症的有用方法，尤其是在大规模研究中。
