Institute of Molecular Immunology, HelmholtzZentrum München, German Research Center for Environmental Health (GmbH), Munich, Germany.
Anal Bioanal Chem. 2012 Jan;402(2):989-95. doi: 10.1007/s00216-011-5475-4. Epub 2011 Oct 18.
Procalcitonin (PCT)-a diagnostic serum parameter for bacterial infection and sepsis-is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC(50)) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7 μg L(-1) (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R (2): assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.
降钙素原 (PCT)-一种用于细菌感染和败血症的诊断血清参数-在即时检测的生物传感器领域引起了极大的兴趣。其检测需要特定的生物识别元件，如抗体。在此，我们描述了用于 PCT 的大鼠单克隆抗体 (mAb) 的开发和表征，以及它们在酶联免疫吸附测定 (ELISA) 中用于测定患者血清样本中 PCT 的应用。从大约 50 个 mAb 中，选择了两个 mAb，CALCA 2F3 和 CALCA 4A6，作为用于夹心免疫测定中 PCT 的高亲和力配对。这两种 mAb 都可以用作捕获或检测 mAb。当用作检测 mAb 时，它们经过 Protein G 纯化和生物素化。使用重组人 (hr) PCT 标准品建立了两种夹心 ELISA，分别使用 CALCA 2F3 (测定 A) 或 CALCA 4A6 (测定 B) 作为捕获 mAb，以及生物素化 mAb CALCA 4A6 或 CALCA 2F3 作为检测 mAb，可特异性测定 PCT，无降钙素和降钙素原的交叉反应。两种测定法的测试中点 (IC(50)) 分别在 4%(w/v) 人血清白蛋白中的 hrPCT 标准品中确定，发现分别为 2.5(测定 A)和 2.7 μg L(-1) (测定 B)。使用这两种夹心 ELISA，与 Elecsys BRAHMS PCT 测定法相比，对 8 个患者血清样本进行了测定。我们的原型 ELISA 与 BRAHMS 测定法之间可以很好地相关 (R(2)：测定 A，0.996 和测定 B，0.990)。这些新开发的抗 PCT mAb 的使用将在即时检测败血症和全身性炎症过程的免疫传感器中找到广泛的应用。
