1. Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol.
- Author
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Ihara M, Suzuki T, Kobayashi N, Goto J, and Ueda H
- Subjects
- Adrenal Cortex Hormones blood, Adrenal Cortex Hormones immunology, Alkaline Phosphatase metabolism, Antibody Specificity, Carrier Proteins genetics, Carrier Proteins metabolism, Cloning, Molecular, Cortodoxone blood, Cortodoxone immunology, Horseradish Peroxidase metabolism, Humans, Immobilized Proteins metabolism, Immunoglobulin Variable Region genetics, Immunoglobulin Variable Region immunology, Immunoglobulin Variable Region metabolism, Limit of Detection, Luminescent Measurements, Maltose-Binding Proteins, Mutation, Peptide Library, Pituitary-Adrenal Function Tests, Protein Stability, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Recombinant Fusion Proteins metabolism, Surface Properties, Adrenal Cortex Hormones analysis, Cortodoxone analysis, Enzyme-Linked Immunosorbent Assay methods
- Abstract
A noncompetitive immunoassay has the potential for improved sensitivity and working range compared with corresponding competitive assays. However, monovalent antigens with less than 1000 in molecular weight are not susceptible to sandwich assays due to their small size. As a noncompetitive immunoassay that can be performed with a clone of an antibody, an open-sandwich immunoassay (OS-IA) based on the antigen-dependent stabilization of the antibody variable region (V(H) + V(L)) was applied to the quantification of 11-deoxycortisol (11-DC; M(r) 346.5), a corticosteroid serving as a diagnostic index for pituitary-adrenal function, as a model target hapten. By one step OS-IA detection of enzyme-labeled V(H) fragment bound to immobilized V(L) in the presence of sample in microplate wells, 11-DC was measured with a femtomolar detection limit and the working range was wider than that with corresponding competitive assay. In addition, the selectivity against analogues was found almost identical to that of conventional assays. The effect of the mutagenesis of a V(H) residue at the V(H)/V(L) interface to reduce background signal was also shown, implying the wider application of OS-IA in small molecule analyses.
- Published
- 2009
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