title: Design of luciferase-displaying protein nanoparticles for use as highly sensitive immunoassay detection probes.
abstract: In this study, we developed a protein nanoparticle-based immunoassay to detect cancer biomarkers using a bioluminescent fusion protein. This method relies on the use of protein nanoparticles comprised of genetically-engineered elastin-like polypeptides (ELPs) fused with poly-aspartic acid tails (ELP-D), previously developed in our lab. The sizes of the self-assembled ELP-D nanoparticles can be regulated at the nanoscale by charged repulsion of the poly-aspartic acid chains. To improve the sensitivity of enzyme-linked immunosorbent assays (ELISAs), we herein demonstrate the multivalent display of NanoLuc® (Nluc) luciferase and a biotin acceptor peptide (BAP) on the surfaces of ELP-D nanoparticles, and demonstrate the sensitivity of these multivalent nanoparticles as detection probes. The fusion protein comprised of ELP-D and Nluc-BAP (ELP-D-Nluc-BAP) was found to form nanoparticles with Nluc and BAP displayed multivalently on their surfaces. Moreover, the use of the nanoparticles in ELISA resulted in a detection sensitivity for α-fetoprotein (AFP) about 10 times higher than that of an assay relying on the use of the monomeric version of the fusion protein. Taken together, ELP-D-based nanoparticles displaying multivalent luciferases on their surfaces enable the construction of an ELISA with enhanced sensitivity.
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan. ekobatak@bio.titech.ac.jp.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan. ekobatak@bio.titech.ac.jp.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mashimo
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan. ekobatak@bio.titech.ac.jp.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Mie
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan. ekobatak@bio.titech.ac.jp.
authors.forename: Eiry
authors.initials: E
authors.lastname: Kobatake
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D001710:Biotin; D004549:Elastin; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay; D008156:Luciferases / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D010455:Peptides
chemical_list: D014408:Biomarkers, Tumor; D010455:Peptides; D001710:Biotin; D004549:Elastin; D008156:Luciferases