Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol.
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.
Ihara
Masaki
M
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Suzuki
Tatsuya
T
Kobayashi
Norihiro
N
Goto
Junichi
J
Ueda
Hiroshi
H
D000305:Adrenal Cortex Hormones / Q000032:analysis* / Q000097:blood / Q000276:immunology; D000469:Alkaline Phosphatase / Q000378:metabolism; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003350:Cortodoxone / Q000032:analysis* / Q000097:blood / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057230:Limit of Detection; D008163:Luminescent Measurements; D058619:Maltose-Binding Proteins; D009154:Mutation; D019151:Peptide Library; D010912:Pituitary-Adrenal Function Tests; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013499:Surface Properties
D000305:Adrenal Cortex Hormones; D002352:Carrier Proteins; D055767:Immobilized Proteins; D007135:Immunoglobulin Variable Region; D058619:Maltose-Binding Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D003350:Cortodoxone