Development of rapid immunoasssay using nanoluc-derived peptide tags
September 24-28, 2017
Conventional immunoassays require multiple steps, and are labor and time-consuming. Hence, rapid and handy immunoassays with high sensitivity are desired. Protein-fragment complementation assay (PCA) of an enzyme such as luciferase will be a promising approach to realize such homogeneous immunoassay. Previously, a luciferase from the deep sea shrimp Oplophorus gracilirostris (NanoLuc, Nluc) was divided into two fragments, SmBit (11 aa) and LgBit (18 kDa) [Fig. 1 left], and each fused with interacting partners . When expressed in mammalian cells, upon interaction, increased luminescence was observed. However, detection of PCA using purified proteins in vitro has been elusive.
The affinity between the two antibody variable region fragments, VL and VH, remarkably increases when antigen binds to them (Open-sandwich principle) . In this study, first, LgBit and SmBit were fused with VH and VL to detect antigen peptide [Fig. 1, left]. Next, LgBit was divided into MidBit and SmBit2 (11 aa), and SmBit and SmBit2 were each fused with VL and VH, respectively, to obtain antigen-dependent luminescent signal upon reconstitution with MidBit [Fig. 1, right].
cDNA for SmBit, SmBit2 and MidBit were synthesized by Eurofin Genomics (Tokyo, Japan). VL and VH fragments of anti-BGP antibody  were each fused with LgBit and SmBit, or fused with SmBit and SmBit2, respectively. They were expressed as a fusion protein with thioredoxin and His6 tag in E. coli SHuffle Express T7 LysY, and purified by TALON immobilized metal affinity resin.
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Yuki Ohmuro-Matsuyama and Hiroshi Ueda, "Development of rapid immunoasssay using nanoluc-derived peptide tags" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/enzyme_xxiv/90