An enzyme-labeled immunometric assay for quantitation of digoxin in serum or plasma.

A heterogeneous enzyme-labeled immunometric assay for quantifying digoxin in serum or plasma was developed. The sample's drug reacts with an excess of an antidigoxin Fab'-beta-galactosidase monoconjugate and then the free monoconjugate is removed by polyacrylamide digitoxigenin-coupled beads; the beta-galactosidase activity of the supernatant measured photometrically at 634 nm is directly proportional to the digoxin concentration in the sample. The assay shares the basic reagents for the immunological reaction with the Seralyzer dry-strip immunometric assay; the reagents for the indicator enzyme reaction were instead formulated in liquid form for use with common clinical analyzers. The test requires a two-point calibration (a 3.0 micrograms/L digoxin calibrator and a reagent blank). One sample can be assayed in approximately 20 min; the assay range is from 0.3 to 5.0 micrograms/L. Dilution tests showed an average found/expected ratio of 100.6%. Mean analytical recovery was 99.2%. Overall coefficients of variation (CVs) (three replicates for 12 runs over 15 days; daily calibration) ranged from 4.9 to 10.0% (Technicon RA-50) and from 2.5 to 10.0% (Cobas Fara) for samples with digoxin concentrations of 4.7 to 0.5 micrograms/L. No interference was found by high levels of common analytes (bilirubin, triglycerides, hemoglobin, total protein, uric acid) or anticoagulants. Cross-reactivity by digoxin metabolites and structurally-related compounds was investigated. Good correlations were found with radiommunoassay (RIA) (r = 0.986) and enzyme immunoassay (EIA) (r = 0.994). Thus, the assay is a specific, reliable, and convenient method for measuring digoxin in both small and large laboratories.

Enzymatic assay of magnesium through glucokinase activation.

We describe an improved enzymatic method for assaying magnesium in serum, plasma, or urine. Magnesium participates as an Mg.ATP complex in a reaction catalyzed by glucokinase (EC 2.7.1.2) coupled to an NADP+-dependent glucose-6-phosphate dehydrogenase (EC 1.1.1.49) reaction. The increase of absorbance at 340 nm, due to the NADPH produced, is proportional to the amount of the activated glucokinase, which in turn is related to the concentration of magnesium in the sample. The method is characterized by a zero-order reaction kinetics, affording a simple and rapid assay with good sensitivity and linearity (up to 2.06 mmol/L) and by working solutions that are stable (refrigerated) for one month. The method is reliable, produces test results that compare closely with those of the atomic absorption spectrophotometry (r greater than or equal to 0.99), is suitable for routine work, and lends itself to automation.