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Development of an immunoassay for endogenous digitalislike factor
D W Harris1, M A Clark, J F Fisher
1Upjohn Laboratories, Upjohn Company, Kalamazoo, MI 49001.
Hypertension (Dallas, Tex. : 1979)
|June 1, 1991
Summary
Researchers developed an enzyme-linked immunosorbent assay (ELISA) to detect endogenous digitalis-like factor (EDLF), a circulating sodium pump inhibitor. This assay confirms EDLF is naturally occurring and plays a role in electrolyte balance.
Area of Science:
- Biochemistry
- Physiology
- Immunology
Background:
- Circulating inhibitors of the sodium pump have been identified.
- Mass spectral analysis guided the purification of these inhibitors.
Purpose of the Study:
- To develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting endogenous digitalis-like factor (EDLF).
- To characterize the specificity and sensitivity of the developed ELISA.
- To measure circulating EDLF levels in humans.
Main Methods:
- Antibodies were raised in rabbits against ouabain conjugates.
- An indirect ELISA was developed using these antibodies.
- Solid-phase extraction and ELISA were used to quantify EDLF in human plasma.
Main Results:
- The developed antisera showed high titer and specificity for ouabain and related cardenolides.
- The ELISA demonstrated a working range of 5-2,000 fmol with an IC50 of 80 fmol/well.
- Circulating EDLF levels were measured in normal volunteers (138 +/- 43 fmol/ml) and patients on total parenteral nutrition (108 +/- 17 fmol/ml).
Conclusions:
- The developed ELISA effectively measures endogenous digitalis-like factor (EDLF).
- EDLF is of endogenous origin and its levels may differ between healthy individuals and those on total parenteral nutrition.
- This assay provides a tool to study the role of EDLF in modulating the sodium pump and cellular electrolyte homeostasis.