Digoxin ELISA
The Quantitative Determination of Digoxin Concentration in Human Serum or Plasma by a Microplate Enzyme Immunoassay
The clinical usefulness of the measurement of serum digoxin (DIG) is due to its low therapeutic ratio; a very small difference exists between therapeutic and toxic tissue levels. In addition, individuals may vary in their response to digoxin with an apparent increase in susceptibility to toxicity with age (1).
The action of digoxin is to increase the force and velocity of myocardial contraction. This is necessary in the treatment of congestive heart failure and arrhythmias such as atrial fibrillation and atrial flutter (2).
The myocardial concentrations of digoxin to serum levels remain relatively constant during normal renal function. This distribution ratio of digoxin is approximately 29 to 1 between the heart and serum (3).
Thus, monitoring digoxin therapy by measurement of serum levels is feasible from the pharmacological standpoint, since serum levels are related to tissue levels following post-absorption equilibration (1). A practical and sensitive method of digoxin quantitation in serum is by enzyme immunoassay.
This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations. In this method, serum reference, patient specimen, or control is first added to a microplate well. Digoxin coupled with enzyme horseradish peroxidase (HRP) is added, and the reactants are mixed. A competition reaction results between the enzyme conjugated digoxin and the native digoxin for a limited number of antibody combining sites immobilized on the well.
After the completion of the required incubation period, the antibody bound enzyme-digoxin conjugate is separated from the unbound enzyme-digoxin conjugate by aspiration or decantation. Excess unbound enzyme reagent is washed via a wash step. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color.
The employment of several serum references of known digoxin concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen’s activity can be correlated with digoxin concentration.
The essential reagents required for a solid phase enzyme immunoassay include immobilized antibody, enzyme-antigen conjugate and native antigen. Upon mixing immobilized antibody, enzyme-antigen conjugate and a serum containing the native antigen, a competition reaction results between the native antigen and the enzyme-antigen conjugate for a limited number of insolubulized binding sites.
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