Human Digoxin ELISA Kit

Sample

Serum, Plasma

Species Reactivity

Human

Detection Method

cELISA

Intended Use

The Quantitative Determination of Digoxin Concentration in Human Serum or Plasma by a Microplate Enzyme Immunoassay, Colorimetric

Contents of Kit

A. Dig Calibrators - 1ml/vial
Six(6) vials of serum reference for digoxin at concentrations of 0 (A), 0.25 (B), 0.5 (C), 1.0 (D),2.0 (E) and 4.0 (F) ng/ml. Store at 2-8°C. A preservative has been added.
B. Dig Enzyme Reagent-6ml/vial
One (1) vial of Digoxin-Horseradish peroxidase (HRP) conjugate in a buffer with dye. A preservative has been added. Store at 2-8°C.
C. Dig Biotin Reagent -6ml/vial
One(1) vial of reagent contains anti-digoxin biotinylated rabbit serum conjugate in buffer, dye and preservative. Store at 2-8°C
D. Streptavidin Coated Plate - 96 wells
One 96-well microplate coated with 1.0 μg/ml streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C
E. Wash Solution Concentrate -20ml/vial
One (1) vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-8°C.
F. Substrate A-7ml/vial
One (1) vial contains tetramethylbenzidine (TMB) in buffer. Store at 2-8°C.
G. Substrate B --7ml/vial
One (1) vial contains hydrogen peroxide (H₂O₂) in buffer. Store at 2-8°C.
H. Stop Solution - 8ml/vial
One (1) vial contains a strong acid(1N HCl). Store at 2-8°C.
I. Product Instructions.

Storage

Note 1: Do not use reagents beyond the kit expiration date.
Note 2: Avoid extended exposure to heat and light. Opened reagents are stable for sixty (60) days when stored at 2-8°C. Kit and component stability are identified on the label.
Note 3: Above reagents are for a single 96-well microplate.

Performance Characteristics

Accuracy:
The DIG ELISA test system was compared against a predicate Digoxin method. Biological specimens from a general population were used. The values ranged from 0.5 -2.917 ng/ml. The correlation is presented in Table.

Only slight amounts of bias between this method and the reference method are indicated by the closeness of the mean values. The least square regression equation and correlation coefficient also indicates excellent method agreement.

Precision

The within and between assay precisions of the DIG ELISA test system were determined by analyses on three different levels of pool control sera. The number (N), mean values (X), standard deviation (s) and coefficient of variation (C.V.) for each of these control sera are presented in Tables.


*As measured in ten experiments in duplicate over a ten day period.

Sensitivity

The DIG ELISA test system has an analytical sensitivity of 0.072 ng/ml. The sensitivity was ascertained by determining the variability of the '0' calibrator and using the 2σ (95% certainty) statistic to calculate the minimum concentration.

General Description

The clinical usefulness of the measurement of serum digoxin 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. 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. 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. Thus, monitoring digoxin therapy by measurement of serum levels is feasible from the pharmacological standpoint, since serum levels are related to tissue levels following postabsorption equilibration. 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 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.