TY - JOUR
T1 - An enzyme-linked immunometric assay for cortisol based on idiotype-anti-idiotype reactions
AU - Niwa, Toshifumi
AU - Kobayashi, Takayuki
AU - Sun, Pi
AU - Goto, Junichi
AU - Oyama, Hiroyuki
AU - Kobayashi, Norihiro
N1 - Funding Information:
Portions of this work were supported by grants from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan. We would like to thank the Health Science Resources Bank (Osaka, Japan) for providing the myeloma P3/NS1/1-Ag-4 cells.
PY - 2009/4/6
Y1 - 2009/4/6
N2 - 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.
AB - 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.
KW - Anti-idiotype antibody
KW - Cortisol
KW - Enzyme-linked immunosorbent assay
KW - Hapten
KW - Monoclonal antibody
KW - Noncompetitive assay
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U2 - 10.1016/j.aca.2009.02.010
DO - 10.1016/j.aca.2009.02.010
M3 - Article
C2 - 19298885
AN - SCOPUS:61949256426
VL - 638
SP - 94
EP - 100
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
SN - 0003-2670
IS - 1
ER -