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 gamma1, kappa) showed an affinity constant (K(a)) for cortisol of 1 x 10(9) 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 microg/day (n = 7) and 80.8 +/- 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.
Kobayashi
Norihiro
N
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
Sun
Pi
P
Fujimaki
Yayoi
Y
Niwa
Toshifumi
T
Nishio
Tadashi
T
Goto
Junichi
J
Hosoda
Hiroshi
H
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001826:Body Fluids / Q000737:chemistry; D002138:Calibration; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D006854:Hydrocortisone / Q000097:blood* / Q000276:immunology / Q000652:urine*; D008297:Male; D051379:Mice; D012710:Serum Albumin, Bovine / Q000276:immunology
D000911:Antibodies, Monoclonal; D012710:Serum Albumin, Bovine; D006854:Hydrocortisone