title: Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method.
abstract: An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10(-19) mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.
authors: BL Co. Ltd., Numazu 410-0042, Japan.
authors: Satoshi
authors: S
authors: Watabe
authors: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki 769-2193, Japan.
authors: Hiromi
authors: H
authors: Kodama
authors: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki 769-2193, Japan.
authors: Mugiho
authors: M
authors: Kaneda
authors: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki 769-2193, Japan; TAUNS Co. Ltd., Izunokuni 410-2325, Japan.
authors: Mika
authors: M
authors: Morikawa
authors: TAUNS Co. Ltd., Izunokuni 410-2325, Japan.
authors: Kazunari
authors: K
authors: Nakaishi
authors: Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan.
authors: Teruki
authors: T
authors: Yoshimura
authors: Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
authors: Atsushi
authors: A
authors: Iwai
authors: Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
authors: Toshiaki
authors: T
authors: Miura
authors: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki 769-2193, Japan.
authors: Etsuro
authors: E
authors: Ito
keywords: 3α-hydroxysteroid dehydrogenase; androsterone; enzyme cycling; insulin; thio-NAD