Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding
| Autor: | Tatsunori Shirai, Masahiro Okumura, Michimasa Kishimoto, Manami Inoue, Yoichi Kumada, Eiju Sasaki, Aya Nakagawa, Kyoto Hamasaki |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Recombinant Fusion Proteins
Immunology Antibody Affinity Enzyme-Linked Immunosorbent Assay Peptide Protein Engineering medicine.disease_cause Protein Refolding Immunoglobulin Fab Fragments Mice chemistry.chemical_compound Phase (matter) Escherichia coli medicine Animals Urea Immunology and Allergy Serologic Tests Volume concentration chemistry.chemical_classification Heavy chain Chromatography biology High-Throughput Screening Assays chemistry biology.protein Polystyrenes Immunoglobulin Light Chains Antibody Immunoglobulin Heavy Chains Peptides Antibodies Immobilized Antibody screening Protein Binding |
| Zdroj: | Journal of Immunological Methods. :70-77 |
| ISSN: | 0022-1759 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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