title: IgG-detection devices for the Tus-Ter-lock immuno-PCR diagnostic platform.
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.
authors: Comparative Genomics Centre, School of Pharmacy & Molecular Sciences, James Cook University, Townsville, QLD, Australia.
authors: Isabelle
authors: I
authors: Morin
authors: Samuel P
authors: SP
authors: Askin
authors: Patrick M
authors: PM
authors: Schaeffer
mesh_terms: D000818:Animals; D000941:Antigens / Q000032:analysis / Q000276:immunology; D001483:Base Sequence; D002645:Chickens; D004247:DNA / Q000235:genetics / Q000378:metabolism; D003955:Diagnostic Tests, Routine / Q000295:instrumentation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D000941:Antigens; D029968:Escherichia coli Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; C489236:tus protein, E coli; D004247:DNA