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 journalArticlepeer-review

33 Citations (Scopus)


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)
Publication statusPublished - 2014
Externally publishedYes


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

ASJC Scopus subject areas

  • Biophysics


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