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

2009; Elsevier BV; Volume: 638; Issue: 1 Linguagem: Inglês

10.1016/j.aca.2009.02.010

1873-4324

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

Receptor Mechanisms and Signaling

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 beta-type and four alpha-type anti-idiotype antibodies, recognizing the paratope and framework regions, respectively. An immunometric assay was established using a combination of a selected alpha-type and a selected beta-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 beta-type antibody. Hapten-occupied anti-cortisol antibody, with less steric hindrance, was then selectively bound by the alpha-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.