Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 1997-05, Vol.43 (5), p.715-722
• Main Authors: Harwood, Steven, Little, John A, Gallacher, Gerard, Perrett, David, Edwards, Raymond, Dawnay, Anne
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid; Fundamental and applied biological sciences. Psychology; Humans; Immunoenzyme Techniques - statistics & numerical data; Microchemistry; Other biological molecules; Ouabain - blood; Quality Control; Reference Values; Sensitivity and Specificity; Terpenes, steroids. Hormones
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/43.5.715

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive-minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)-and has a working range (between-assay CV < 10%) of 180-10000 pmol/L (54-3000 pmol/L in plasma). Mean recoveries of ouabain added to plasma ranged from 90% to 100%, and plasma extracts diluted in parallel to the standard curve. Plasma OLC concentrations in 10 healthy volunteers averaged 92 pmol/L (range 55-168), assuming 100% cross-reactivity of OLC in the ouabain assay. HPLC analysis with two distinct chromatographic conditions demonstrated that endogenous human plasma OLC co-eluted with authentic ouabain. The enzyme immunoassay is rapid and easy to perform and will support further investigation of the nature of this controversial endogenous steroid.