An enzyme-linked immunometric assay for cortisol based on idiotype-anti-idiotype reactions

Toshifumi Niwa, Takayuki Kobayashi, Pi Sun, Junichi Goto, Hiroyuki Oyama, Norihiro Kobayashi

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)


Cortisol levels in body fluids are useful for monitoring the function of the pituitary-adrenal axis. Here, we established an "enzyme-linked immunometric assay" (a noncompetitive-type ELISA) for cortisol based on idiotype-anti-idiotype reactions. Six different anti-idiotype monoclonal antibodies that recognized the variable regions of a newly established anti-cortisol antibody were generated using hybridoma technology; these were two β-type and four α-type anti-idiotype antibodies, recognizing the paratope and framework regions, respectively. An immunometric assay was established using a combination of a selected α-type and a selected β-type antibody. The analyte (cortisol) was captured by an excess amount of anti-cortisol antibody immobilized on microplates, and the unoccupied paratope was saturated with the β-type antibody. Hapten-occupied anti-cortisol antibody, with less steric hindrance, was then selectively bound by the α-type antibody, labeled with biotin. The amount of biotin residue on the microplates was colorimetrically monitored using a peroxidase-labeled streptavidin. This assay had an approximately threefold higher sensitivity (detection limit: 90 pg = 248 fmol cortisol) than a competitive ELISA using the same anti-cortisol antibody, as well as a practical specificity for providing reasonable determination of normal urinary cortisol levels.

Original languageEnglish
Pages (from-to)94-100
Number of pages7
JournalAnalytica Chimica Acta
Issue number1
Publication statusPublished - 2009 Apr 6
Externally publishedYes


  • Anti-idiotype antibody
  • Cortisol
  • Enzyme-linked immunosorbent assay
  • Hapten
  • Monoclonal antibody
  • Noncompetitive assay

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy


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