Hypertension, Vol 17, 936-943, Copyright © 1991 by American Heart Association
DW Harris, MA Clark, JF Fisher, JM Hamlyn, KP Kolbasa, JH Ludens and DW DuCharme
Recently, attempts to purify and identify a circulating inhibitor of the
sodium pump have been successful. Based on the outcome of mass spectral
analysis of purified inhibitor, we raised in rabbits antibodies to
conjugates of the commercially available cardenolide ouabain and used them
in the development of an indirect enzyme-linked immunosorbent assay (ELISA)
for endogenous digitalislike factor (EDLF). Antisera obtained were of high
antibody titer (1:2 x 10(6)) and showed full cross-reactivity with purified
EDLF. The antisera were highly specific for ouabain and structurally
related cardenolides but showed no cross-reactivity with numerous
endogenous steroids and peptides. At each step in the purification of EDLF,
inhibition of the sodium pump and immunologic cross-reactivity were
inseparable. The ELISA as developed had a working range of 5-2,000 fmol,
with an IC50 of 80 fmol/well. Using solid-phase extraction and the ELISA,
we determined the circulating level of EDLF in plasma from normal human
volunteers to be 138 +/- 43 fmol/ml, whereas patients on total parenteral
nutrition for at least 1 week had a circulating level of 108 +/- 17
fmol/ml, suggesting that the circulating factor was of endogenous origin.
The ELISA developed appears to measure a naturally occurring counterpart to
the cardenolides that could play a role in modulating the sodium pump and
thereby cellular electrolyte homeostasis.
ARTICLES
Development of an immunoassay for endogenous digitalislike factor
Upjohn Laboratories, Upjohn Company, Kalamazoo, MI 49001.
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