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

Research output: Contribution to journalArticle

12 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 languageEnglish
Pages (from-to)49-54
Number of pages6
JournalBiophysics (Japan)
Volume10
DOIs
Publication statusPublished - 2014
Externally publishedYes

Fingerprint

Hydroxysteroids
NAD
3-Hydroxysteroid Dehydrogenases
Enzyme-Linked Immunosorbent Assay
Ketosteroids
Proteins
Androsterone
Antibodies
Alkaline Phosphatase
Hydrolysis
Phosphates
Antigens
thionicotinamide adenine dinucleotide

Keywords

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

ASJC Scopus subject areas

  • Biophysics

Cite this

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method. / Watabe, Satoshi; Kodama, Hiromi; Kaneda, Mugiho; Morikawa, Mika; Nakaishi, Kazunari; Yoshimura, Teruki; Iwai, Atsushi; Miura, Toshiaki; Ito, Etsuro.

In: Biophysics (Japan), Vol. 10, 2014, p. 49-54.

Research output: Contribution to journalArticle

Watabe, S, Kodama, H, Kaneda, M, Morikawa, M, Nakaishi, K, Yoshimura, T, Iwai, A, Miura, T & Ito, E 2014, 'Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method', Biophysics (Japan), vol. 10, pp. 49-54. https://doi.org/10.2142/biophysics.10.49
Watabe, Satoshi ; Kodama, Hiromi ; Kaneda, Mugiho ; Morikawa, Mika ; Nakaishi, Kazunari ; Yoshimura, Teruki ; Iwai, Atsushi ; Miura, Toshiaki ; Ito, Etsuro. / Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method. In: Biophysics (Japan). 2014 ; Vol. 10. pp. 49-54.
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