Abstract
Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which 10Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.
| Original language | English |
|---|---|
| Pages (from-to) | 288-299 |
| Number of pages | 12 |
| Journal | Journal of Biotechnology |
| Volume | 127 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jan 1 2007 |
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Keywords
- Affinity peptide
- Hydrophilic polystyrene surface
- Immunoassay
- One-step ELISA
- PS-tag
ASJC Scopus subject areas
- Biotechnology
Cite this
Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface. / Kumada, Yoichi; Katoh, Shigeo; Imanaka, Hiroyuki; Imamura, Koreyoshi; Nakanishi, Kazuhiro.
In: Journal of Biotechnology, Vol. 127, No. 2, 01.01.2007, p. 288-299.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface
AU - Kumada, Yoichi
AU - Katoh, Shigeo
AU - Imanaka, Hiroyuki
AU - Imamura, Koreyoshi
AU - Nakanishi, Kazuhiro
PY - 2007/1/1
Y1 - 2007/1/1
N2 - Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which 10Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.
AB - Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which 10Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.
KW - Affinity peptide
KW - Hydrophilic polystyrene surface
KW - Immunoassay
KW - One-step ELISA
KW - PS-tag
UR - http://www.scopus.com/inward/record.url?scp=33846255071&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33846255071&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2006.07.011
DO - 10.1016/j.jbiotec.2006.07.011
M3 - Article
VL - 127
SP - 288
EP - 299
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 2
ER -