Immunoenzymometric assay for a small molecule, 11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies

Norihiro Kobayashi, Keiichi Iwakami, Shuhei Kotoshiba, Toshifumi Niwa, Yoshinori Kato, Nariyasu Mano, Junichi Goto

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

Abstract

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 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.

Original languageEnglish
Pages (from-to)2244-2253
Number of pages10
JournalAnalytical Chemistry
Volume78
Issue number7
DOIs
Publication statusPublished - 2006 Apr 1

ASJC Scopus subject areas

  • Analytical Chemistry

Fingerprint Dive into the research topics of 'Immunoenzymometric assay for a small molecule, 11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies'. Together they form a unique fingerprint.

Cite this