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Hiroyuki Oyama, Eri Tanaka, Tomoyo Kawanaka, Izumi Morita, Toshifumi Niwa, and Norihiro Kobayashi.

Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.

Hiroyuki Oyama, Izumi Morita, Yuki Kiguchi, Sayaka Miyake, Ayaka Moriuchi, Tatsuki Akisada, Toshifumi Niwa, and Norihiro Kobayashi.

Journal of the American Chemical Society 2022, 144 Antibody–Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers. Leonard, Miguel Aller Pellitero, Boris Juelg, Jamie B. This article is cited by 37 publications. The plasma 11-DC levels determined for healthy subjects were validated as reliable.

This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity.

As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. 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. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. 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. 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.

We selected 11-deoxycortisol (11-DC M r 346.5), a corticosteroid serving a diagnostic index for pituitary−adrenal function, as a model target hapten. 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.