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

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 (K_a) 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.