Immunoenzymometric Assay for a Small Molecule,11-Deoxycortisol, with Attomole-Range Sensitivity Employing an scFv−Enzyme Fusion Protein and Anti-Idiotype Antibodies
| Autor: | Junichi Goto, Nariyasu Mano, Keiichi Iwakami, Toshifumi Niwa, Yoshinori Kato, Norihiro Kobayashi, Shuhei Kotoshiba |
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| Rok vydání: | 2006 |
| Předmět: |
Cortodoxone
Molecular Sequence Data Immunoglobulin Variable Region Pituitary-Adrenal System Enzyme-Linked Immunosorbent Assay chemical and pharmacologic phenomena Analytical Chemistry Mice Microtiter plate Antibody Specificity Animals Polyacrylamide gel electrophoresis Base Sequence Dose-Response Relationship Drug biology Chemistry Fusion protein Small molecule Antibodies Anti-Idiotypic Spectrometry Fluorescence Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Alkaline phosphatase Electrophoresis Polyacrylamide Gel Paratope Antibody Haptens Hapten |
| Zdroj: | Analytical Chemistry. 78:2244-2253 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac051858f |
| Popis: | To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable. |
| Databáze: | OpenAIRE |
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