Comparative Genomics Centre, School of Pharmacy & Molecular Sciences, James Cook University, Townsville, QLD, Australia.
Analyst. 2011 Nov 21;136(22):4815-21. doi: 10.1039/c1an15731k. Epub 2011 Oct 6.
The number of new Immuno-PCR technologies and applications is steadily growing as a result of a general need for more sensitive immunoassays for early detection of diseases. Although Immuno-PCR has been demonstrated to be superior to its immunoassay counterpart, it is still regarded as a challenging technology due to various problems arising from its increased detection power, such as high background noise as well as substantial batch-to-batch reproducibility issues. Current efforts have intensified to produce homogeneous universal protein-DNA conjugates to simplify this technology and render it more robust. We have recently developed a new quantitative Immuno-PCR (qIPCR) technology using the Tus-Ter-lock (TT-lock) interaction to produce homogeneous protein-DNA conjugates that can detect very small numbers of disease-related antibodies. We now report the further development of the TT-lock Immuno-PCR platform for the quasi universal quantitative detection of antigens and mammalian IgG. For this, Tus was fused to various IgG-binding proteins--i.e. protein G, protein L and their LG chimera--and self-assembled to the TT-lock-T template. These detection devices were then evaluated and applied in various direct and indirect Immuno-PCR formats. The direct TT-lock qIPCR could detect goat anti-GFP IgG at concentrations as low as 0.3 pM and total human IgG in serum samples with great sensitivity. Further indirect TT-lock qIPCR systems were developed that could detect 1 pM of GFP and 10 pM of measles nucleoprotein. In all cases, the superiority of the TT-lock Immuno-PCR was demonstrated in terms of sensitivity over an analogous Protein G-Peroxidase ELISA.
由于需要更灵敏的免疫分析方法来早期检测疾病，新型免疫 PCR 技术和应用的数量正在稳步增长。尽管免疫 PCR 已被证明优于其免疫分析对应物，但由于其检测能力的提高所带来的各种问题，例如高背景噪声和大量批间重现性问题，它仍然被认为是一项具有挑战性的技术。目前，人们正在加紧努力生产均质通用的蛋白质-DNA 缀合物，以简化该技术并使其更具鲁棒性。我们最近开发了一种新的定量免疫 PCR（qIPCR）技术，该技术使用 Tus-Ter-lock（TT-lock）相互作用来产生均质的蛋白质-DNA 缀合物，从而可以检测到非常少量的与疾病相关的抗体。我们现在报告了 TT-lock Immuno-PCR 平台的进一步发展，用于抗原和哺乳动物 IgG 的准通用定量检测。为此，将 Tus 融合到各种 IgG 结合蛋白中，即蛋白 G、蛋白 L 和它们的 LG 嵌合体，并自组装到 TT-lock-T 模板上。然后评估和应用了这些检测装置在各种直接和间接免疫 PCR 格式中。直接 TT-lock qIPCR 可以检测到浓度低至 0.3 pM 的山羊抗 GFP IgG 和血清样品中的总人 IgG，具有很高的灵敏度。进一步开发了间接 TT-lock qIPCR 系统，可以检测到 1 pM 的 GFP 和 10 pM 的麻疹核蛋白。在所有情况下，与类似的蛋白 G-过氧化物酶 ELISA 相比，TT-lock Immuno-PCR 在灵敏度方面都表现出优越性。
