title: Improved lectin ELISA for glycosylation analysis of biomarkers using PS-tag-fused single-chain Fv.
abstract: In this study, we successfully developed a novel lectin enzyme-linked immunosorbent assay (lectin ELISA) for the detection of glycosides linked to carcinoembryonic antigen (CEA) as a model antigen using a scFv-immobilized hydrophilic polystyrene (phi-PS) plate and 12 different HRP-labeled lectins. Anti-CEA scFv genetically fused with a PS-tag at its C-terminus (scFv-PS) was successfully over-expressed in an insoluble fraction by cultivation of recombinant E. coli. A solid-phase refolding method was adopted to immobilize and refold scFv-PS on the surface of the phi-PS plate. Consequently, in sandwich ELISA using phi-PS plates immobilized with scFv and scFv-PS as well as Maxisorp™ plate with whole antibody (whole Ab), the highest sensitivity and S/N ratio were obtained from the scFv-PS-immobilized phi-PS plate as it was the antigen-binding domain with the highest surface immobilization density and remaining activity displayed on the phi-PS surface. During the lectin ELISA, high background signals were detected from the whole-Ab-immobilized Maxisorp™ plate, indicating that HRP-labeled lectins, particularly PHA-E, Con A, LCA, PSA, DSA, and MAA, directly recognized the glyco-chains of a whole Ab. However, a considerable amount of low background signals were detected from the scFv-PS-immobilized plate since the ligand antibody, namely scFv-PS, was produced by E. coli cells that had no potential for glycosylation during the process of post-transcriptional modification. Signals for glyco-chains conjugated with CEA were detectable using 6 kinds of HRP-labeled lectins, namely PHA-E, PHA-L, SBA, Con A, LCA, and DSA, with much higher sensitivities and S/N ratios. Thus, the lectin ELISA using scFv-PS as a ligand was considerably useful for detecting the glyco-chains of glycoproteins, including glyco-biomarkers, with much higher sensitivities and S/N ratios compared with a conventional whole Ab. By preparation of a phi-PS plate immobilized with different species of scFvs, this method is capable of the glyco-chain analysis of a number of glyco-biomarkers in the fields of clinical diagnosis and biochemical research.
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, Japan. kumada@kit.ac.jp
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kumada
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ohigashi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Emori
authors.forename: Koji
authors.initials: K
authors.lastname: Imamura
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Omura
authors.forename: Michimasa
authors.initials: M
authors.lastname: Kishimoto
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000276:immunology; D055910:Antibodies, Immobilized / Q000276:immunology; D015415:Biomarkers / Q000032:analysis* / Q000378:metabolism; D002272:Carcinoembryonic Antigen / Q000032:analysis / Q000235:genetics / Q000276:immunology; D003208:Concanavalin A / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D018796:Immunoconjugates / Q000235:genetics / Q000276:immunology; D037102:Lectins / Q000276:immunology*; D010835:Phytohemagglutinins / Q000276:immunology; D011137:Polystyrenes / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D012685:Sepharose / Q000031:analogs & derivatives; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D000906:Antibodies; D055910:Antibodies, Immobilized; D015415:Biomarkers; D002272:Carcinoembryonic Antigen; D018796:Immunoconjugates; D037102:Lectins; D010835:Phytohemagglutinins; D011137:Polystyrenes; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C045779:concanavalin A-horseradish peroxidase conjugate; C028326:concanavalin A-sepharose; C067494:erythroagglutinating phytohemagglutinin; D003208:Concanavalin A; D012685:Sepharose; D006735:Horseradish Peroxidase