Development of Lectin Immunoassay for Detection of Glyco-Chain Conjugated with Biomarker

Recently, it has been clarified that various glycol-chains with special structures are conjugated with protein biomarkers according to appearance and/or proceeding of serious diseases in human body. Therefore, sensitive detection methods for not only amount of biomarker itself, but also its glycol-chain structure are highly requisite in the field of clinical diagnosis. In this study, we developed a novel lectin immunoassay method that special glyco-form structures conjugated could be specifically detected with high sensitivity and S/N ratio using single-chain Fv (scFv) antibody and HRP-labeled lectin. Polystyrene-binding peptide (PS-tag: RIIIRRIRR) which was originally isolated in our research group, was genetically-fused at the C-terminal region of scFv (30kDa) for site-specific attachment with higher density and maintenance of higher antigen-binding activity, compared with the conventional whole antibody (whole Ab, 150kDa). PS-tag-fused scFv (scFv-PS) was over-expressed in E. coli, and it was site-specifically immobilized on the surface of polystyrene (PS) plate. When the scFv-immobilized PS plate was used in sandwich ELISA using carcinoembryonic antigen (CEA) as a model glycobiomarker, more than 50-fold higher sensitivity was detected, compared with the conventional PS plate immobilized with whole Ab. Lectin immunoassay using the scFv-immobilized PS plate and 12 types of HRP-labeld lectins allowed detection of signals on the basis of the specific interaction between lectin and glyco-chain conjugated. Much higher signal / noise (S/N) ratios were obtained from scFv-immobilized PS plate in the lectin immunoassay, while higher background signals detected from whole Ab-immobilized PS plate significantly decreased S/N ratios, because lectins strongly bound to the whole Ab which was also a kinds of glycoproteins. Thus, the lectin immunoassay using scFv-immobilized PS plate developed in this study is considerably useful for detection of a variety of glyco-forms conjugated with protein biomarkers.