Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface.
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 (10)Lys 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.
Kumada
Yoichi
Y
Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan.
Katoh
Shigeo
S
Imanaka
Hiroyuki
H
Imamura
Koreyoshi
K
Nakanishi
Kazuhiro
K
D000345:Affinity Labels; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001120:Arginine / Q000235:genetics; D001667:Binding, Competitive; D019921:Biotinylation; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation* / Q000379:methods*; D005982:Glutathione Transferase / Q000032:analysis; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000032:analysis; D008024:Ligands; D008239:Lysine / Q000235:genetics; D010455:Peptides / Q000378:metabolism*; D011137:Polystyrenes / Q000737:chemistry*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D000345:Affinity Labels; D000906:Antibodies; D000941:Antigens; D007328:Insulin; D008024:Ligands; D010455:Peptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D012710:Serum Albumin, Bovine; D019809:Streptavidin; D001120:Arginine; D005982:Glutathione Transferase; D008239:Lysine