Abstract
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.