Design and site-directed immobilization of single-chain Fv antibody to polystyrene latex beads via material-binding peptides and application to latex turbidimetric assay
| Autor: | Koichi Takahashi, Reina Tanibata, Yohei Miyamura, Jun-ichi Horiuchi, Yoichi Kumada, Fumio Gondaira, Shinya Ogasawara |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Latex chemical and pharmacologic phenomena Bioengineering Peptide Applied Microbiology and Biotechnology 01 natural sciences 03 medical and health sciences chemistry.chemical_compound Antigen 010608 biotechnology Reactivity (chemistry) chemistry.chemical_classification Latex beads Chromatography biology Chemistry respiratory system Ligand (biochemistry) Microspheres 030104 developmental biology biology.protein Polystyrenes Polystyrene Antibody Peptides Antibodies Immobilized Single-Chain Antibodies Biotechnology |
| Zdroj: | Journal of Bioscience and Bioengineering. 131:84-89 |
| ISSN: | 1389-1723 |
| Popis: | In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm3, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm2, which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads. |
| Databáze: | OpenAIRE |
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