Analytical Sciences

Abstract − Analytical Sciences, 18(12), 1309 (2002).

Generation of a Novel Monoclonal Antibody against Cortisol-[C-4]-Bovine Serum Albumin Conjugate: Application to Enzyme-Linked Immunosorbent Assay for Urinary and Serum Cortisol
Norihiro KOBAYASHI,* Pi SUN,* Yayoi FUJIMAKI,* Toshifumi NIWA,** Tadashi NISHIO,* Junichi GOTO,*  and Hiroshi HOSODA***
*Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan
**Department of Medical Technology, College of Medical Sciences, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
***Daiichi Pure Chemicals Co., Ltd., 3-13-5, Nihombashi, Chuo-ku, Tokyo 103-0027, Japan
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 gamma 1, kappa) showed an affinity constant (Ka) for cortisol of 1 x 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%), 17alpha-hydroxyprogesterone (12%), 6beta-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 ug/day (n = 7) and 80.8 ± 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.