A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins
A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins
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DOI：
10.1016/s0006-291x(03)01364-0
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发表时间：
2003-08-22
影响因子：
3.1
通讯作者：
Niemeyer, CM
中科院分区：
文献类型：
--
作者：
Adler, M;Wacker, R;Niemeyer, CM
采用标准化的自组装dna -蛋白偶联物作为试剂，结合高度敏感的免疫PCR （IPCR）和基于TaqMan原理的成熟、可靠、快速的实时PCR检测方法，建立了一种快速、可靠的检测方法。使用抗物种免疫球蛋白试剂可以很容易地适应这种分析基本上任何现有的ELISA应用。在实时IPCR （rtIPCR）中使用内部竞争对手进一步提高了该检测的敏感性和重要性；采用直接、间接和夹心模型rtIPCR法检测多种物种（小鼠、兔、山羊和人）的0.1-0.01 amol (500-50 fg/mL) IgG，从而将类似ELISA检测的检出限提高约100- 1000倍。通过检测人血浆样品中40 pg/mL的新型抗癌药物rViscumin和细胞培养基中100 pg/mL的研究抗体，证明了该方法的稳健性。在这两种情况下，可比ELISA的灵敏度都要低1000倍。(C) 2003 Elsevier Inc.版权所有。
A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive. (C) 2003 Elsevier Inc. All rights reserved.