Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Satoshi Watabe; Hiromi Kodama; Mugiho Kaneda; Mika Morikawa; Kazunari Nakaishi; Teruki Yoshimura; Atsushi Iwai; Toshiaki Miura; Etsuro Ito

doi:10.2142/biophysics.10.49

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Satoshi Watabe, Hiromi Kodama, Mugiho Kaneda, Mika Morikawa, Kazunari Nakaishi, Teruki Yoshimura, Atsushi Iwai, Toshiaki Miura, Etsuro Ito^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

32 Citations (Scopus)

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.

Original language	English
Pages (from-to)	49-54
Number of pages	6
Journal	Biophysics (Japan)
Volume	10
DOIs	https://doi.org/10.2142/biophysics.10.49
Publication status	Published - 2014
Externally published	Yes

Keywords

3α-hydroxysteroid dehydrogenase
Androsterone
Enzyme cycling
Insulin
Thio-NAD

ASJC Scopus subject areas

Biophysics

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10.2142/biophysics.10.49

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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.",

keywords = "3α-hydroxysteroid dehydrogenase, Androsterone, Enzyme cycling, Insulin, Thio-NAD",

author = "Satoshi Watabe and Hiromi Kodama and Mugiho Kaneda and Mika Morikawa and Kazunari Nakaishi and Teruki Yoshimura and Atsushi Iwai and Toshiaki Miura and Etsuro Ito",

note = "Publisher Copyright: {\textcopyright} 2014 THE BIOPHYSICAL SOCIETY OF JAPAN.",

year = "2014",

doi = "10.2142/biophysics.10.49",

language = "English",

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T1 - Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

AU - Watabe, Satoshi

AU - Kodama, Hiromi

AU - Kaneda, Mugiho

AU - Morikawa, Mika

AU - Nakaishi, Kazunari

AU - Yoshimura, Teruki

AU - Iwai, Atsushi

AU - Miura, Toshiaki

AU - Ito, Etsuro

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - 3α-hydroxysteroid dehydrogenase

KW - Androsterone

KW - Enzyme cycling

KW - Insulin

KW - Thio-NAD

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ER -

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Abstract

Keywords

ASJC Scopus subject areas

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