Immunoenzymometric Assay for a Small Molecule,11-Deoxycortisol, with Attomole-Range Sensitivity Employing an scFv−Enzyme Fusion Protein and Anti-Idiotype Antibodies
2006; American Chemical Society; Volume: 78; Issue: 7 Linguagem: Inglês
10.1021/ac051858f
ISSN1520-6882
AutoresNorihiro Kobayashi, Keiichi Iwakami, Shuhei Kotoshiba, Toshimitsu Niwa, Yoshinori Katō, Nariyasu Mano, Junichi Goto,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoTo 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 β-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 α-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.
Referência(s)