Adler Michael, Wacker Ron, Niemeyer Christof M
Chimera Biotec GmbH, Emil-Figge-Str. 76 A, D-44227 Dortmund, Germany.
Biochem Biophys Res Commun. 2003 Aug 22;308(2):240-50. doi: 10.1016/s0006-291x(03)01364-0.
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.
本文介绍了一种快速且稳健的检测方法，该方法基于高灵敏度免疫PCR（IPCR）与通过TaqMan原理进行的成熟、可靠且快速的实时PCR检测相结合，其中IPCR采用标准化的自组装DNA-蛋白质缀合物作为试剂。使用抗物种免疫球蛋白试剂可使该检测方法轻松适用于基本上任何现有的ELISA应用。在实时IPCR（rtIPCR）中使用内部竞争物进一步提高了该检测方法的灵敏度和重要性；使用直接、间接和夹心模型rtIPCR检测方法可检测到来自几种物种（小鼠、兔子、山羊和人类）的0.1 - 0.01 amol（500 - 50 fg/mL）IgG，从而使类似ELISA检测的检测限提高了约100至1000倍。通过在人类血浆样本中检测40 pg/mL的新型抗癌药物天花粉蛋白以及在细胞培养基中检测100 pg/mL的研究性抗体，在两个典型应用中证明了该方法的稳健性。在这两种情况下，可比的ELISA灵敏度低1000倍。
