title: Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.
abstract: In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 606-8585, Japan. Electronic address: kumada@kit.ac.jp.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kumada
authors.forename: Kyoto
authors.initials: K
authors.lastname: Hamasaki
authors.forename: Aya
authors.initials: A
authors.lastname: Nakagawa
authors.forename: Eiju
authors.initials: E
authors.lastname: Sasaki
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Shirai
authors.forename: Masahiro
authors.initials: M
authors.lastname: Okumura
authors.forename: Manami
authors.initials: M
authors.lastname: Inoue
authors.forename: Michimasa
authors.initials: M
authors.lastname: Kishimoto
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry*; D000915:Antibody Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D057166:High-Throughput Screening Assays; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011137:Polystyrenes / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012698:Serologic Tests / Q000379:methods; D014508:Urea / Q000737:chemistry
chemical_list: D055910:Antibodies, Immobilized; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D010455:Peptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D014508:Urea
keywords: ELISA; Fab antibody; Immobilization; Polystyrene-binding peptide (PS-tag); Solid-phase refolding