Digoxin Immunoassays Research Articles

Digoxin immunoassay with cross-reactivity of digoxin metabolites proportional to their biological activity

Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.

Homogeneous Amperometric Immunoassay for Digoxin

Abstract A homogeneous spectrophotometric EMIT immunoassay for the measurement of digoxin has been converted to an amperometric immunoassay format capable of analysing digoxin in diluted serum. NADH produced by glucose-6-phosphate dehydrogenase (G6PDH) labelled digoxin was detected amperometrically by its rapid, efficient oxidation at platinized activated carbon electrodes (PACE) poised at +150 mV vs Ag/AgCl. The new amperometric procedure resulted in a simplified assay protocol with a decreased overall assay time, enhanced NADH detection and minimal sample pretreatment.

Characterization of digoxigenin-labeled B-phycoerythrin by capillary electrophoresis with laser-induced fluorescence Application to homogeneous digoxin immunoassay

A laser-induced fluorescence (LIF) immunoassay technique based on capillary electrophoretic (CE) separation is demonstrated. The analysis of digoxin in serum at clinically useful concentration levels of 10 −9 to 10 −10 M is achieved using this technique. The chemistry presented here using digoxigenin-labeled B-phycoerythrin was selected as a convenient model for the exploration of CE-LIF-based immunoassays. The LIF system described here exhibits detection limits in the low 10 −11 M range for several common fluorophores. The data presented in this report are one of the first examples of nanomolar quantitative analysis in a human serum matrix by CE.

Immunological detection of cardiac glycosides in plants

Australian native and introduced plants were examined, using digoxin immunoassays, to detect the presence of cross-reacting cardiac glycosides. These compounds were found in 27 species from 20 genera. The assay technique can also be used on serum samples to confirm cardiac glycoside ingestion.

Specificity of digoxin immunoassays toward digoxin metabolites

Specificity of digoxin immunoassays toward digoxin metabolites

Di-2-ethylhexyl phthalate: A medical concern and a possible marker for chronic fatigue syndrome

Abstract

Digoxin immunoassay and Chinese medicine

Digoxin immunoassay and Chinese medicine

The Extent of Cross-Reactivity with Digoxin-Like Immunore-active Factors (DLIFS) Investigated with Two Homogeneous Immunoassays for Digoxin

s Third International Congress of Therapeutic Drug Monitoring and Clinical Toxicology: PDF Only

Cross-Reactivity of Anti-Digoxin Antibodies with Digitoxin Depends on Tracer Structure

The most common methods for measuring digoxin concentrations in serum are immunoassays. The prerequisite for exact determination of the digoxin value is an antibody that specifically binds digoxin. Because digitoxin differs from digoxin only in the C-12 hydroxy group, it is difficult to obtain anti-digoxin antibodies that do not cross-react with this compound. During the development of a fluorescence polarization immunoassay (FPIA) for digoxin, we investigated digoxin tracers with different structures. We found that in FPIA the digitoxin cross-reactivity of an antibody could be reduced by varying the structure of the tracer molecule.

The contribution of steroids to digoxin-like immunoreactivity in cord blood.

Samples of cord serum from 29 healthy neonates were analysed for digoxin-like immunoreactive substance (DLIS), cortisol, 17 beta-oestradiol, progesterone, dehydroepiandrosterone-sulphate (DHEAS), 17 alpha-hydroxyprogesterone (17OHP), androstenedione, oestriol and ouabain-like activity (OLA; by inhibition of Na+, K+ATPase activity). The mean serum concentration of DLIS was 0.91 (SD = 0.19) nmol/L and the mean OLA was 26.1 (SD = 11.5) nmol/L. There was no correlation between DLIS and OLA. DLIS correlated significantly with oestriol (r = 0.521), progesterone (r = 0.534) and 17OHP (r = 0.43). Stepwise multiple regression analysis showed that 17 beta-oestradiol, progesterone and androstenedione contributed to DLIS and the intercept was 0.64 (SD = 0.127). The concentrations of steroids (17 beta-oestradiol, progesterone, androstenedione) required to displace digoxin by 50% in the digoxin immunoassay and inhibit Na+,K+ATPase in the OLA assay were 10(3)-10(4)-fold higher than those found in cord serum. We conclude that the contribution of these steroids to DLIS is small and that DLIS and OLA measure different compounds.

Determination of free serum digoxin concentrations in digoxin toxic patients after administration of digoxin fab antibodies.

Digoxin fab antibody therapy is known to interfere with digoxin immunoassays causing spurious serum digoxin concentrations. The reliability and precision of three digoxin immunoassays--Baxter Dade Stratus (BDS), Syva affinity column enzyme-mediated immunoassay (EMIT), and the reference assay Abbott TDx fluorescence polarization immunoassay following ultrafiltration (FPIA-UF)--were compared in eight digoxin toxic patients treated with digoxin fab antibodies. Five to eight blood samples were drawn serially up to 204 h post digoxin fab therapy. The serum digoxin concentration in each sample was determined by each of the three assays. The mean (+/- SD) area under the serum digoxin concentration-time curve was significantly lower for FPIA-UF than for BDS or EMIT (86.1 +/- 58.2 vs 158.1 +/- 88.6 and 176.3 +/- 115.3 h.ng/ml p less than 0.01, respectively). BDS correlated better with FPIA-UF (r2 = 0.71) than did EMIT (r2 = 0.45). Predictive performance of the BDS and EMIT assays demonstrated that the mean prediction error (bias) (0.62 vs 0.78 ng/ml) and the mean squared prediction error (precision) (0.48 vs 0.76) differed significantly from zero (p less than 0.05). However, BDS had significantly less bias and greater precision than did EMIT (p less than 0.05). In the presence of digoxin fab antibodies, BDS is a better predictor of free serum digoxin concentration than is EMIT, but both have considerable bias. Based on these results, FPIA-UF should be the assay of choice for determining free serum digoxin concentrations during fab therapy.

058 Endogenous digoxin-like immunoreactive factors (DLIF): Impact on the results of two homogeneous immunoassays for digoxin

058 Endogenous digoxin-like immunoreactive factors (DLIF): Impact on the results of two homogeneous immunoassays for digoxin

Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics

Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene.

Aluminum phthalocyanine-streptavidin: new, sensitive fluorescent tracer for immunoassay

A new fluorescent reagent based on aluminum phthalocyanine has been developed for general application to immunoassay. This highly sensitive fluorophore, Ultralite 680, may be covalently attached to a variety of biological entities for use as a tracer. We report the use of an Ultralite 680-streptavidin-digoxin conjugate as a tracer in a direct, competitive, heterogeneous, fluorescent immunoassay of digoxin. The assay is performed manually with 50 microL of serum and no sample pre-treatment. Primary anti-digoxin antibody is incubated with a serum sample containing digoxin and Ultralite 680-streptavidin-digoxin. Bound and free tracer are separated, and the bound fluorescence is released and quantified with a commercially available spectrofluorometer. Assay precision was good (CV = 7.05% at a digoxin concentration of 2.00 micrograms/L) and results by our method correlated well with those by a conventional RIA (r = 0.9650, n = 24).

Novel Anti-Digoxin Monoclonal Antibodies with Different Binding Specificities for Digoxin Metabolites and Other Glycosides

Spleen cells of BALB/c mice immunized with a digoxin-bovine serum albumin conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Seven monoclonal antibodies (MAb) selected by indirect ELISA were produced, purified and characterized. All the MAb were IgG1 isotypes. The apparent equilibrium association constants (Ka) of four of the MAb, determined by Scatchard analysis of the RIA data, ranged from 1 x 10(9) M-1 to 5.9 x 10(9) M-1. The estimated Ka values of the three other MAb were found to be between 4.8 x 10(7) M-1 and 5.9 x 10(8) M-1. Using digoxin and eighteen structurally-related compounds, the seven MAb could be divided into five groups based on their binding specificities assessed by an inhibition immunoenzymatic test. The MAb in Groups I and II, in particular, showed very different specificity profiles: the two MAb in Group I had low cross-reactivity with cardioinactive digoxin metabolites, whereas the high affinity MAb in Group II recognized all the digoxin metabolites tested. The MAb in Group I might be useful in a digoxin immunoassay and the Group II MAb in therapeutic reversal of digoxin intoxication.

Effect of Digoxin Fab Antibodies on Five Digoxin Immunoassays

Following digoxin Fab antibody (FAB) administration in digitalis-toxic patients, total serum digoxin concentrations (SDCS) become elevated, but do not correlate with pharmacologic activity. In an attempt to accurately measure free (pharmacologically active) SDC in the presence of FAB, we assessed the utility of five digoxin immunoassays: fluorescence polarization immunoassay (FPIA), ultrafiltration with FPIA (ULTRA-FPIA), enzyme multiplied immunoassay (EMIT), radioimmunoassay (RIA), and American Dade's STRATUS (STRATUS). To normal human serum samples containing 2 and 4 ng/ml of digoxin, FAB was added in escalating quantities of 0-1.9 micrograms. In addition, 1.9 micrograms of FAB was added to two serum samples containing no digoxin. SDCS reported by FPIA for each 2 ng/ml samples, in order of ascending FAB doses, were 2.08, 1.94, 2.02, 1.99, 1.95, and 1.93 ng/ml, while the SDCS from the ULTRA-FPIA were 2.00, 1.76, 1.56, 1.36, 1.16, and 0.98 ng/ml. Results similar to the ULTRA-FPIA were obtained with the STRATUS, RIA, and EMIT, although the SDCS from the EMIT (p less than 0.05) samples exhibited greater fluctuation. The 4 ng/ml samples demonstrated similar patterns among the assays although no statistical differences were noticed between EMIT and ULTRA-FPIA. Samples containing FAB without digoxin only adversely affected the RIA, which reported mean SDCS from the two identically prepared samples of 5.8 and 7.8 ng/ml. Except for the FPIA, the SDC measured by the assays directly correlated with the amount of FAB in the sample, demonstrating the ability of these assays to measure free SDC.

A study of the CEDIA digoxin immunoassay

We evaluated the CEDIA digoxin immunoassay (Microgenics, Inc., Concord, CA), as performed with the Cobas-Bio centrifugal analyzer. In assays of sera with known concentrations of digoxin, the enzyme activity, measured when two beta-galactosidase (EC 3.2.1.23) fragments were combined according to the assay format, was proportional to the digoxin concentration. Results of assays of sera containing 0 to 3 micrograms of digoxin per liter correlated well when compared with an RIA method: CEDIA, microgram/L = 1.00 x RIA - 0.06 microgram/L (n = 90, r = 0.95, Sxy = 0.05). Duplicate assays of three control sera containing 0.8, 2.2, or 3.3 micrograms/L, each analyzed 20 times a day with each group of patients' samples, gave within-run CVs of 1-3% and day-to-day CVs of 3-12%. The reconstituted CEDIA reagents were stable for at least a month at 5 degrees C. As many as 25 samples and controls can be assayed in half the time needed to complete a similar number of RIA measurements with comparable results.

Digoxin-like Immunoreactive Substances in the Plasma of Intensive Care Unit Patients: Relationship to Organ Dysfunction

Digoxin-like immunoreactive substances are an endogenous group of compounds that cross-react in conventional immunoassays for digoxin. Plasma digoxin-like immunoreactive substance concentrations were estimated using the Abbott TDxll fluorescence polarisation immunoassay kit for digoxin. Digoxin-like immunoreactive substances were measured in one hundred consecutive Intensive Care Unit (ICU) patients who were not treated with digoxin. One hundred healthy blood donors were used as controls. Thirty of the ICU patients had plasma digoxin-like immunoreactive substance concentrations greater than or equal to the greatest value found in the control group (0.22 nmol/l). In the ICU group the median value was 0.17 nmol/l and the range zero to 1.69 nmol/l. In the control group the median was less than the limit of detection of the assay, and the range zero to 0.22 nmol/l. Sixteen ICU patients had coexisting renal and hepatic dysfunction and this group had a median digoxin-like immunoreactive substance concentration of 0.21 nmol/l (range zero to 1.69 nmol/l), while 38 patients with hepatic dysfunction and normal renal function had a median concentration of 0.17 nmol/l (range zero to 0.77 nmol/l). In contrast four patients with renal dysfunction only had a median concentration of 0.05 nmol/l (range zero to 0.34 nmol/l). The remaining forty-two patients had neither hepatic nor renal dysfunction and this group had a median concentration of 0.15 nmol/l (range zero to 0.36 nmol/l). This study has identified the critically ill as a group of patients who exhibit measurable plasma digoxin-like immunoreactive substances using the most commonly used kit for analysis of digoxin.

Digoxin Immunoassay with Monoclonal and Polyclonal Antibodies Using Time-Resolved Fluorometry

We describe a nonisotopic heterogeneous competitive immunoassay of digoxin in serum using either Fab fragments of a polyclonal antibody or a high-affinity monoclonal antibody. In the assay, digoxin competes with immobilized digoxin (digoxin:thyroglobulin conjugate) for binding to a biotinylated immunoreactant (Fab or monoclonal). The amount of biotinylated moiety bound to the solid phase (white polystyrene microtiter wells), which is inversely related to the amount of digoxin in the sample, is then quantified by adding streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The fluorescent immunocomplex formed is measured directly on the dry solid phase by time-resolved fluorometry. The assay is simple to perform and its characteristics are similar to those of other currently used immunoassay techniques. The Fab fragments and the monoclonal antibody procedure performed equally well on the system. Our results suggest that monoclonal antibodies with high affinity for digoxin can be used for the routine determination of the drug in serum.

Hydrophilic fluorescein derivatives: Useful reagents for liposome immunolytic assays

Hydrophilic derivatives of fluorescein containing hydroxyalkyl substituents were synthesized and encapsulated within liposomes. The fluorophores showed significantly more retention with time than did fluorescein, carboxyfluorescein, or calcein. Unlike calcein, the fluorophores are minimally susceptible to fluorescence quenching by Co 2+ The utility of these compounds as immunodiagnostic reagents was demonstrated by encapsulating 5(6)-carboxyfluorescein trismethylolamide in haptenized liposomes which were used in an immunoassay for digoxin.