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Clinical Chemistry 43: 715-722, 1997;
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(Clinical Chemistry. 1997;43:715-722.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

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

Steven Harwood1, John A. Little3, Gerard Gallacher1, David Perrett2, Raymond Edwards3 and Anne Dawnay1,a

1 Department of Clinical Biochemistry and
2 Department of Medicine, Renal Research Laboratory, St. Bartholomew's Hospital Centre for Clinical Research, London ECIA 7BE, UK.

3 Netria, Department of Chemical Endocrinology, St. Bartholomew's Hospital, London EC1A 7BE, UK.
a Author for correspondence. Fax 44-171-796-4676; e-mail A.B.Dawnay{at}mds.qmw.ac.uk

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 180–10 000 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.


Key Words: indexing terms: chromatography, reversed-phase • antibody specificity




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Copyright © 1997 by the American Association for Clinical Chemistry.