Detection of small molecule diagnostic markers with phage-based open-sandwich immuno-PCR

Open sandwich immunoassay (OS-IA) utilizes antigen-dependent stabilization of antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. To further improve its detection sensitivity, here we employed phage-based immuno-PCR approach. wherein OS-IA and quantitative PCR methodologies were combined with the use of immobilized V-L fusion protein and filamentous phages displaying V-H fragment, whose DNA was extracted for PCR amplification. This approach significantly enhanced the assay sensitivity for small molecule antigens osteocalcin (BGP) peptide and 17beta-estradiol. (C) 2012 Elsevier B.V. All rights reserved.