Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers

Abstract

Open sandwich (OS) immunoassay utilizes antigen-dependent stabilization of an antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. For further improvement of its sensitivity, OS Immuno-PCR was attempted with recombinant fusion proteins. The maltose binding protein-fused heavy chain variable region (MBP-V(H)) of an antibody that recognizes the C-terminal fragment of human osteocalcin (bone Gla protein, BGP), a biomarker for bone-related diseases, was immobilized onto microplate wells, and the antigen together with streptavidin (SA)-fused light chain variable region of the same antibody (SA-V(L)) was added and incubated. The amount of immobilized SA-V(L) was quantified by tethered biotinylated DNA, which was used to estimate the amount of antigen by realtime PCR. When BGP C-terminal peptide was detected, the limit of detection was 100 fg/mL, which was superior than that of our previously reported phage-based OS Immuno-PCR. The developed OS Immuno-PCR system will be useful for the detection of small molecule biomarkers for disease prevention.

Sensitive Ligand-Based Protein Quantification Using Immuno-PCR: A Critical Review Of Single-Probe And Proximity Ligation Assays

“…In this sense, sandwich is perhaps a slight misnomer. Hasan et al brought iPCR into OS-ELISA by engineering a streptavidin and single-chain variable fragment (scFv) fusion protein to target osteocalcin (41). Tethering of the stabilized complex to the wells of an ELISA plate was mediated by maltose binding protein fused with V H , and PCR-compatibility was made possible by the biotinylated DNA binding properties of streptavidin.…”

Mimetic peptide and special antibody: promising agents for optimizing hapten-analyzing systems

“…Moreover, phages should be freshly prepared, which can affect the reproducibility and practical utility of the assay. To eliminate all of these limitations, Hasan et al 61 conducted further efforts to develop a streptavidin (SA)-based OS-IPCR system for the detection of BGP-C7. The MBP-fused V H was immobilized onto microplate wells, and BGP-C7 together with SA-fused V L (SA-V L ) was added and incubated.…”