An enzyme-linked immunometric assay for cortisol based on idiotype-anti-idiotype reactions.
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.
Niwa
Toshifumi
T
Department of Medical Technology, Health Sciences, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Kobayashi
Takayuki
T
Sun
Pi
P
Goto
Junichi
J
Oyama
Hiroyuki
H
Kobayashi
Norihiro
N
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D006854:Hydrocortisone / Q000276:immunology* / Q000378:metabolism / Q000652:urine; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007130:Immunoglobulin Idiotypes; D006854:Hydrocortisone