Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women.
We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.
Fiet
Jean
J
Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France. jean.fiet@libertysurf.fr
Giton
Frank
F
Auzerie
Jack
J
Galons
Hervé
H
D002715:Chlormadinone Acetate / Q000008:administration & dosage / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D003280:Contraceptives, Oral, Synthetic / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics; D015914:Estrogen Replacement Therapy; D005260:Female; D015200:Fluoroimmunoassay / Q000379:methods* / Q000592:standards; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008593:Menopause / Q000097:blood* / Q000187:drug effects; D015394:Molecular Structure; D045167:Progesterone Congeners / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D003280:Contraceptives, Oral, Synthetic; D007106:Immune Sera; D045167:Progesterone Congeners; D002715:Chlormadinone Acetate