TY - JOUR
T1 - Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate
T2 - Application to enzyme-linked immunosorbent assay for urinary and serum cortisol
AU - Kobayashi, Norihiro
AU - Sun, Pi
AU - Fujimaki, Yayoi
AU - Niwa, Toshifumi
AU - Nishio, Tadashi
AU - Goto, Junichi
AU - Hosoda, Hiroshi
PY - 2002/12/1
Y1 - 2002/12/1
N2 - Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype γl, κ) showed an affinity constant (Ka) for cortisol of 1 × 109 M-1 and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17α-hydroxyprogesterone (12%), 6β-hydroxycortisol (8.4%), and tetrahydrocortisol (<0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 ± 17.0 μg/day (n = 7) and 80.8 ± 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.
AB - Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype γl, κ) showed an affinity constant (Ka) for cortisol of 1 × 109 M-1 and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17α-hydroxyprogesterone (12%), 6β-hydroxycortisol (8.4%), and tetrahydrocortisol (<0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 ± 17.0 μg/day (n = 7) and 80.8 ± 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.
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U2 - 10.2116/analsci.18.1309
DO - 10.2116/analsci.18.1309
M3 - Article
C2 - 12502080
AN - SCOPUS:0036952645
VL - 18
SP - 1309
EP - 1314
JO - Analytical Sciences
JF - Analytical Sciences
SN - 0910-6340
IS - 12
ER -