Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface

Yoichi Kumada, Shigeo Katoh, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

Research output: Contribution to journalArticle

35 Citations (Scopus)

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 languageEnglish
Pages (from-to)288-299
Number of pages12
JournalJournal of Biotechnology
Volume127
Issue number2
DOIs
Publication statusPublished - Jan 1 2007

Fingerprint

Immunosorbents
Polystyrenes
Peptides
Assays
Enzymes
Enzyme-Linked Immunosorbent Assay
Proteins
Ligands
Immunosorbent Techniques
Streptavidin
Insulin
Glutaral
Antigens
Glutathione Transferase
Antibodies
Washing
Immobilization
Immunoglobulin G
Rabbits
Molecules

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 journalArticle

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