A new strategy for the development of monoclonal antibodies for the determination of human procalcitonin in serum samples
A new strategy for the development of monoclonal antibodies for the determination of human procalcitonin in serum samples
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DOI：
10.1007/s00216-011-5475-4
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发表时间：
2011
影响因子：
4.3
通讯作者：
E. Kremmer;K. Meyer;F. Grässer;A. Flatley;Miwako Kösters;P. Luppa;P. Krämer
中科院分区：
文献类型：
--
作者：
E. Kremmer;K. Meyer;F. Grässer;A. Flatley;Miwako Kösters;P. Luppa;P. Krämer
降钙素原（PCT）-细菌感染和脓毒症的诊断血清参数-在用于即时检测的生物传感器领域引起了极大的兴趣。其检测需要特异性的生物识别元件，如抗体。在此，我们描述了大鼠PCT单克隆抗体（mAb）的开发和表征，以及它们在酶联免疫吸附试验（ELISA）中的应用，用于测定患者血清样品中的PCT。从约50个mAb中，选择两个mAb，CALCA 2F 3和CALCA 4A 6，作为夹心免疫测定中对PCT具有高亲和力的一对。两种mAb均可用作捕获mAb或检测mAb。当用作检测mAb时，将其进行蛋白G纯化和生物素化。使用CALCA 2F 3（试验A）或CALCA 4A 6（试验B）作为捕获mAb，分别使用生物素化mAb CALCA 4A 6或CALCA 2F 3作为检测mAb，使用人重组（hr）PCT标准品建立两个夹心ELISA，可高度特异性测定PCT，与降钙素和katacalcin无交叉反应性。在4%（w/v）人血清白蛋白中测定hrPCT标准品的两种试验的试验中点（IC 50），结果为2.5（试验A）和2.7 μg L−1（试验B）。与Elecsys BRAHMS PCT测定相比，使用两种夹心ELISA测定了8份患者血清样本。可以证明我们的原型ELISA和BRAHMS测定之间的良好相关性（R2：测定A，0.996和测定B，0.990）。这些新开发的抗PCT mAb的使用应该在用于脓毒症和全身炎症过程的即时诊断的免疫传感器中找到广泛的应用。
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 (IC50) 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 (R2: 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.