Anti-digoxin Antibody Research Articles

Anti-digoxin polyclonal antibody* is beneficial for the treatment of pre-eclampsia,

Anti-digoxin polyclonal antibody* is beneficial for the treatment of pre-eclampsia,

Ouabain-Sensitive α1 Na,K-ATPase Enhances Natriuretic Response to Saline Load

Na,K-ATPase is ubiquitously expressed and is essential for maintaining electrochemical and osmotic gradients. The alpha subunit of Na,K-ATPase is the receptor for cardiotonic steroids, which act through the ouabain-binding site and are important in cardiovascular regulation. Interestingly, the presence of endogenous Na,K-ATPase ligands has been implicated in the natriuretic response to perturbations such as hypertension and salt loading; therefore, it is important to characterize the role of the ouabain-binding sites in this context. Because the alpha1 isoform of mice and rats is relatively ouabain resistant, gene-targeting strategies were used to produce mice with reversed responses of the alpha1 and/or alpha2 isoforms to ouabain to assess for altered natriuretic responses to acute salt loading. Regardless of the sensitivity of the alpha2 isoform to ouabain, conferring ouabain sensitivity to alpha1 augmented the natriuretic response to an acute salt load. In addition, when endogenous Na,K-ATPase inhibitors were sequestered with an anti-digoxin antibody fragment, the sodium excretion rates in the ouabain-sensitive alpha1 isoform mice were equivalent to the ouabain-resistant alpha1 isoform mice. These data suggest that the ouabain-binding site of the alpha1 Na,K-ATPase can participate in the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands.

Engineered Bacterial Outer Membrane Vesicles with Enhanced Functionality

We have engineered bacterial outer membrane vesicles (OMVs) with dramatically enhanced functionality by fusing several heterologous proteins to the vesicle-associated toxin ClyA of Escherichia coli. Similar to native unfused ClyA, chimeric ClyA fusion proteins were found localized in bacterial OMVs and retained activity of the fusion partners, demonstrating for the first time that ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs. For instance, fusions of ClyA to the enzymes β-lactamase and organophosphorus hydrolase resulted in synthetic OMVs that were capable of hydrolyzing β-lactam antibiotics and paraoxon, respectively. Similarly, expression of an anti-digoxin single-chain Fv antibody fragment fused to the C terminus of ClyA resulted in designer “immuno-MVs” that could bind tightly and specifically to the antibody's cognate antigen. Finally, OMVs displaying green fluorescent protein fused to the C terminus of ClyA were highly fluorescent and, as a result of this new functionality, could be easily tracked during vesicle interaction with human epithelial cells. We expect that the relative plasticity exhibited by ClyA as a fusion partner should prove useful for: (i) further mechanistic studies to identify the vesiculation machinery that regulates OMV secretion and to map the intracellular routing of ClyA-containing OMVs during invasion of host cells; and (ii) biotechnology applications such as surface display of proteins and delivery of biologics.

Effect of Osmolytes on the Conformational Stability of Mouse Monoclonal Antidigoxin Antibody in Long-Term Storage

The natural selection of small organic molecules (osmolytes) that protect cellular proteins against denaturing stresses is characteristic of plants and animals that have adapted to environmental extremes. In this study the role of osmolytes on the structural stability of antibody in long-term storage was investigated. Native antibody preparations isolated from cell culture supernatant were formulated with different additives for storage stabilization. The additives studied were arginine, proline, and sorbitol. Structural stability of antibody investigated and effect of these osmolytes on retaining the native structure in long-term storage were established. CD spectra of native IgG showed the characteristics expected for beta-proteins. After the passing of given time, the beta-sheet structure of IgG was reduced but the addition of the osmolyte to antibody solution prevented loosing the ordered structures. These results can be effective for stabilization of proteins in commercial and industrial processes.

Prediction of calibration curves in ria of digoxin: Influence of some variables

Background Competitive protein binding radioimmunoassay (CPB-RIA) is a principal method for quantifying serum digoxin concentration. The accuracy of this method is critically dependent on factors that influence the substitution reaction between unlabelled (Q) antigen (digoxin) with 125I-labelled antigen (M) bound to anti-digoxin antibody (P). We studied the influence of initial concentration of M, ionic strength, and viscosity on the substitution reaction between M and Q. In addition, we propose a kinetic model for this reaction. Methods We used a commercially available CPB-RIA for digoxin, a gamma counter, and a viscosimeter to study the effect of initial concentration of M, ionic strength, viscosity, and temperature on the substitution reaction between M and Q. Data were analyzed using Statistica software. Results The apparent rate constant for the reaction between M and Q in the formation of PM is dependent on the initial concentration of M, and the ionic strength, viscosity, and temperature of the reaction medium, and independent of the concentration of Q. Conclusion A kinetic model for the displacement of the 125I-digoxin by the digoxin in its union to a specific antibody is proposed. Such model adjusts satisfactorily to the results and allows the prediction of the calibration curves of RIA (activity bound to the antibody vs. concentration of digoxin) showing the influence of the concentration of both species, the time of incubation, the viscosity and the ionic strength of the medium, on the sensitivity of the method of RIA on which the analytical determination of the digoxin is based.

Effect of Indian Ayurvedic Medicine Ashwagandha on Measurement of Serum Digoxin and 11 Commonly Monitored Drugs Using Immunoassays: Study of Protein Binding and Interaction With Digibind

Ashwagandha, a popular Ayurvedic medicine, is now available in the United States. Alkaloids found in this herb have structural similarity with digoxin. To study potential interference of Ashwagandha with serum digoxin measurement by immunoassays. Potential interference was also investigated with immunoassays for 11 other commonly monitored drugs. In addition, interaction of components of Ashwagandha with the Fab fragment of antidigoxin antibody (Digibind) was investigated. Two different brands of liquid extract and 1 dry powdered form of Ashwagandha were used for this investigation. Aliquots of drug-free serum were supplemented with various concentrations of Ashwagandha and apparent digoxin concentrations were measured by 3 digoxin immunoassays. Mice were fed with Ashwagandha and apparent digoxin concentrations were measured 1 and 3 hours after feeding. Potential interference of Ashwagandha with immunoassays of 11 other drugs was also investigated. Interaction of components of Ashwagandha with Digibind was studied in vitro. Significant apparent digoxin concentrations were observed both in vitro and in vivo using the fluorescence polarization immunoassay of digoxin, whereas the Beckman and the microparticle enzyme immunoassay digoxin assay demonstrated minimal interference. Immunoassays of 11 other drugs tested were unaffected. When Ashwagandha extract was added to a serum pool containing digoxin, falsely elevated digoxin value was observed with fluorescence polarization immunoassay, but values were falsely lowered when measured by the microparticle enzyme immunoassay. Digibind neutralized digoxin-like immunoreactive components of Ashwagandha in vitro. Components of Ashwagandha interfered with serum digoxin measurements using immunoassays. Digibind neutralized free digoxin-like immunoreactive components of Ashwagandha.

Effect of Indian Ayurvedic medicine Ashwagandha on measurement of serum digoxin and 11 commonly monitored drugs using immunoassays: study of protein binding and interaction with Digibind.

● Context.—Ashwagandha, a popular Ayurvedic medicine, is now available in the United States. Alkaloids found in this herb have structural similarity with digoxin. Objective.—To study potential interference of Ashwagandha with serum digoxin measurement by immunoassays. Potential interference was also investigated with immunoassays for 11 other commonly monitored drugs. In addition, interaction of components of Ashwagandha with the Fab fragment of antidigoxin antibody (Digibind) was investigated. Design.—Two different brands of liquid extract and 1 dry powdered form of Ashwagandha were used for this investigation. Aliquots of drug-free serum were supplemented with various concentrations of Ashwagandha and apparent digoxin concentrations were measured by 3 digoxin immunoassays. Mice were fed with Ashwagandha and apparent digoxin concentrations were measured 1 and 3 hours after feeding. Potential interference of Ashwagandha with immunoassays of 11 other drugs was also investigated. Interaction of components of Ashwagandha with Digibind was studied in vitro. Results.—Significant apparent digoxin concentrations were observed both in vitro and in vivo using the fluorescence polarization immunoassay of digoxin, whereas the Beckman and the microparticle enzyme immunoassay digoxin assay demonstrated minimal interference. Immunoassays of 11 other drugs tested were unaffected. When Ashwagandha extract was added to a serum pool containing digoxin, falsely elevated digoxin value was observed with fluorescence polarization immunoassay, but values were falsely lowered when measured by the microparticle enzyme immunoassay. Digibind neutralized digoxin-like immunoreactive components of Ashwagandha in vitro. Conclusions.—Components of Ashwagandha interfered with serum digoxin measurements using immunoassays. Digibind neutralized free digoxin-like immunoreactive components of Ashwagandha. (Arch Pathol Lab Med. 2007;131:1298–1303)

Digoxin-Like Immunoreactive Factor in Human Cerebrospinal Fluid

Back to table of contents Previous article Next article LETTERFull AccessDigoxin-Like Immunoreactive Factor in Human Cerebrospinal FluidRif S. El-Mallakh M.D.James Miller Ph.D.Roland Valdes Jr., Ph.D.,Tamella Buss Cassis M.D.,Rena Li M.D., Ph.D.,Rif S. El-Mallakh M.D.Search for more papers by this authorJames Miller Ph.D.Search for more papers by this authorRoland Valdes Jr., Ph.D.Search for more papers by this author,Tamella Buss Cassis M.D.Search for more papers by this author,Rena Li M.D., Ph.D.Search for more papers by this author,Published Online:1 Jan 2007AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail SIR: Mammals produce a family of endogenous compounds that resemble the sodium pump-inhibiting cardiac glycosides, digoxin and ouabain. 1 These endogenous cardenolides have been proposed to be important in the pathophysiology of bipolar illness, where it is relatively deficient in the serum of manic subjects. 1 The measurement of these compounds in the CNS has not been previously documented. Refrigerated cerebrospinal fluid (CSF) waste was frozen at −70°C within 18 hours of collection. Digoxin-like immunoreactive factor (DLIF) was measured by radioimmunoassay as described by Grider et al., 1 using components provided by New England Nuclear (Billerica, Mass.). The correlation among four samples quantified twice was 0.88. Ten samples were studied from three women and seven men. Mean age among the women was 41.9 years (range=30 to 53), and among the men it was 61.6 years (range=32 to 94). Mean DLIF concentration was 91.7 pg digoxin equivalents/ml.Comment Human CSF contains a substance which cross reacts with antidigoxin antibodies. This supports previous reports of CSF factors that bear immunologic resemblance to cardiac glycosides 2 and sodium pump inhibitory activity of CSF. 3 Though the source of CSF DLIF is not known, similar compounds have been purified from mammalian hypothalamus. 4 Given that the hypothalamic-pituitary-adrenal axis may be dysregulated in bipolar illness, that DLIF production is responsive to dexamethasone and adreno-corticotropin hormone (ACTH), 5 and that DLIF may be dysregulated in bipolar subjects, 1 it would seem important to study CSF DLIF in bipolar illness. Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, Ky.Advanced Dermatology and Dermaesthetics, Louisville, Ky.Sun Health Institute, Sun City, Ariz.

Combined use of plasmapheresis and antidigoxin antibodies in a patient with severe digoxin intoxication and acute renal failure

Keywords: digitalis intoxication; renal failure;plasmapheresisDigitalis intoxication is a common and potentiallylife-threatening situation [1]. In this condition, theadministration of digoxin-specific antibodies (Fab)have proven to be effective in controlling seriouscomplications [2]. However, in patients with severerenal impairment, the clearance of Fab–digoxincomplexes is compromised, and this may reduce theutility of Fab therapy in those conditions.Plasmapheresis (PE), by removing the digoxin–Fabcomplexes, was suggested to prevent the phenomenonof rebound toxicity, which is frequently observed inconditions of renal failure [3], and the combined use ofFab and PE in digitalis intoxication associated withsevere renal failure has been reported [4]. However,because in those cases PE was initiated several hoursafter the administration of Fab, the best therapeuticstrategy still remains to be established [5]. We report acase of combined use of Fab closely followed by asingle session of PE in a patient with severe digitalisintoxication.

Homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin employing Ru(bpy)2(dcbpy)NHS and carrier protein

A highly sensitive homogeneous electrogenerated chemiluminescence (ECL) immunoassay for the determination of anti-digoxin antibody and digoxin hapten was developed employing Ru(bpy)(2)(dcbpy)NHS (bpy = 2,2'-bipyridyl; dcbpy = 2,2'-bipyridine-4,4'-dicarboxylic acid; NHS = N-hydroxysuccinimide ester) as an electrochemiluminescent label and bovine serum albumin (BSA) as a carrier protein. A digoxin hapten was indirectly heavily labelled with Ru(bpy)(2)(dcbpy)NHS through BSA to form Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate. The ECL intensity of the immunocomplex of the conjugate with anti-digoxin antibody markedly decreased when the immunoreaction between Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate and anti-digoxin antibody took place. Two formats, direct homogeneous immunoassay for anti-digoxin antibody and competitive immunoassay for digoxin, were developed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration in the range 7.6 x 10(-8)-7.6 x 10(-6) g/mL was determined by direct homogeneous format. Digoxin hapten was determined throughout the range 4.0 x 10(-10)-1.0 x 10(-7) g/mL with a detection limit of 1.0 x 10(-10) g/mL by competitive format. The relative standard derivation for 6.0 x 10(-9) g/mL was 4.3%. The method has been applied to assaying digoxin in control human serum.

Analysis of ligand binding by bioaffinity mass spectrometry

Background Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach. Methods BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between − 91 and − 10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS. Results A protein with 141 kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at ∼ 66 kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with ∼ 3 kDa to ∼ 30 kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts. Conclusions Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.

Therapeutic Drug Monitoring of Digoxin

Digoxin is a cardioactive drug with a narrow therapeutic range. Therapeutic drug monitoring is essential in clinical practice for efficacy as well as to avoid digoxin toxicity. Immunoassays are commonly used in clinical laboratories for determination of serum or plasma digoxin concentrations. Unfortunately, digoxin immunoassays are affected by both endogenous and exogenous compounds. Endogenous compounds are termed 'digoxin-like immunoreactive substances' (DLIS), which are found in elevated concentrations in volume-expanded patients. Exogenous compounds that interfere with digoxin assays are various drugs such as spironolactone, potassium canrenoate as well as Digibind (Fab fragment of antidigoxin antibody), which is used in treating life-threatening digoxin overdose. Moreover, various Chinese medicines such as Chan Su, Lu-Shen Wan and oleander-containing herbal preparations also interfere with serum digoxin measurements by immunoassays. Monitoring unbound (free) digoxin concentration may under certain circumstances eliminate such interferences. Clinicians should be aware of limitations of therapeutic drug monitoring of digoxin using immunoassays.

Effect of Brazilian, Indian, Siberian, Asian, and North American Ginseng on Serum Digoxin Measurement by Immunoassays and Binding of Digoxin-like Immunoreactive Components of Ginseng With Fab Fragment of Antidigoxin Antibody (Digibind)

Effect of Brazilian, Indian, Siberian, Asian, and North American Ginseng on Serum Digoxin Measurement by Immunoassays and Binding of Digoxin-like Immunoreactive Components of Ginseng With Fab Fragment of Antidigoxin Antibody (Digibind)

Effect of Brazilian, Indian, Siberian, Asian, and North American Ginseng on Serum Digoxin Measurement by Immunoassays and Binding of Digoxin-like Immunoreactive Components of Ginseng With Fab Fragment of Antidigoxin Antibody (Digibind)

We compared Brazilian, Indian, Siberian, Asian, and North American ginseng for potential interference with 3 digoxin immunoassays: fluorescence polarization (FPIA), microparticle enzyme (MEIA), and Tina-quant (Roche Diagnostics, Indianapolis, IN). We supplemented aliquots of a drug-free serum pool with ginseng extracts representing expected in vivo concentrations and overdose. We observed apparent digoxin-like immunoreactivity with FPIA, modest immunoreactivity with MEIA, and no apparent digoxin immunoreactivity with the Tina-quant with all ginsengs except Brazilian, which showed no immunoreactivity with any assay. When aliquots of serum pools prepared from patients receiving digoxin were supplemented with ginsengs, we observed falsely elevated digoxin values with FPIA, falsely lower digoxin values (negative interference) with MEIA, and no interference with the Tina-quant. Digoxin-like immunoreactive components of various ginsengs have moderate protein binding; monitoring free digoxin concentrations does not eliminate such interference. We also observed that Digibind (Burroughs Wellcome, Research Triangle Park, NC) can bind free digoxin-like immunoreactive components of ginsengs; such effects can be monitored by measuring apparent free digoxin concentrations. Indian, Asian, and North American ginsengs interfere with serum digoxin measurement by FPIA and MEIA; the Tina-quant is free of such interference. Digibind can bind free digoxin-like immunoreactive components of ginseng.

Molecular modeling of cardiac glycoside binding by the human sequence monoclonal antibody 1B3

The amino acid sequences of the heavy- and light-chain variable regions of the high-affinity human sequence antidigoxin monoclonal antibody 1B3 (mAb 1B3) were determined, and a structural model for the mAb's variable region was developed by homology modeling techniques. The structural model provided the basis for computationally docking digoxin and eight related cardiac glycosides into the putative binding site of mAb 1B3. Analysis of the consensus binding mode obtained for digoxin showed that the cardenolide moiety of digoxin is deeply embedded in a predominantly hydrophobic, narrow cavity, whereas the terminal, gamma-carbohydrate group is solvent-exposed. The docking results indicated that the primary driving forces for digoxin binding by mAb 1B3 are hydrophobic interactions with the digoxin steroid ring system and hydrogen bonds with the digitoxose groups. The binding model accounts for the experimentally observed variations in mAb 1B3 binding affinity for various structural analogs of digoxin used previously to develop a 3D structure-activity relationship model of drug binding (Farr CD, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC, Welsh WJ. Three-dimensional quantitative structure-activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal antibodies using comparative molecular field analysis. J Med Chem 2002;45:3257-3270). In particular, the hydrogen bond pattern is consistent with the unique sensitivity of mAb 1B3's binding affinity to the number of sugar residues present in a cardiac glycoside. The hydrophobic environment about the steroid moiety of digoxin is compatible with the mAb's reduced affinity for ligands that possess hydrophilic hydroxyl and acetyl group modifications in this region. The model also indicated that most of the amino acid residues in contact with the ligand reside in or about the three complementarity determining regions (CDRs) of the heavy chain and the third CDR of the light chain. A comparison of the 1B3 binding model with the crystal structures of two murine antidigoxin mAbs revealed similar binding patterns used by the three mAbs, such as a high frequency of occurrence of aromatic, hydrophobic residues in the CDRs and a dominant role of the heavy chain CDR3 in antigen binding.

New Enzyme-Linked Immunosorbent Digoxin Assay on the ADVIA?? IMS??? 800i System Is Virtually Free from Interference of Endogenous Digoxin-like Immunoreactive Factors

Endogenous digoxin-like immunoreactive factors (DLIF) may crossreact with antidigoxin antibody and falsely elevate immunoassay results. Recently, a new enzyme-linked immunosorbent chemiluminescent assay for digoxin has been available for use on the ADVIA IMS (Integrated Modular System) 800i analyzer (Bayer Diagnostics). We studied potential interference of DLIF with this new digoxin assay. We analyzed 30 serum specimens from patients who have pathologic conditions that may increase serum DLIF concentrations. These patients were never exposed to digoxin or other agents that may lead to a measurable digoxin concentration. We also analyzed 10 specimens from neonates, 10 cord blood specimens, and 10 amniotic fluid specimens. Apparent digoxin concentrations were measured using the new enzyme-linked immunosorbent digoxin assay (IMS-Digoxin), a fluorescence polarization immunoassay (FPIA), and also a chemiluminescent immunoassay (CLIA, run on ACS:180(R) system from Bayer Diagnostics). We observed measurable apparent digoxin levels with the FPIA in 4 uremic patients (range 0.21-0.36 ng/mL, digoxin equivalent), 7 patients with liver disease (range 0.21-0.72 ng/mL), and 3 patients in the third trimester of pregnancy (0.22-0.66 ng/mL). We also observed measurable DLIF concentrations with the FPIA in 2 neonates (0.22 and 0.36 ng/mL), 5 cord blood specimens (range 0.21-1.18 ng/mL), and 5 amniotic fluid specimens (0.21-0.50 ng/mL). None of these DLIF-positive specimens showed any measurable digoxin concentration using the IMS-Digoxin or the CLIA assay. When serum specimens containing elevated concentrations of DLIF but no digoxin (as measured by FPIA) were supplemented with known concentrations of digoxin, we observed falsely elevated digoxin concentrations, as expected, only by the FPIA. In contrast, we observed a good agreement between the target and observed concentrations when the new IMS-Digoxin or the CLIA assay was used. We conclude that the IMS-Digoxin assay is free from interference of DLIF.

Neutralization of Free Digoxin-like Immunoreactive Components of Oriental Medicines Dan Shen and Lu-Shen-Wan by the Fab Fragment of Antidigoxin Antibody (Digibind)

Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro.

Neutralization of Free Digoxin-like Immunoreactive Components of Oriental Medicines Dan Shen and Lu-Shen-Wan by the Fab Fragment of Antidigoxin Antibody (Digibind)

Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro.

Magnetic Separation Immunoassay for Digoxin in Plasma with Flow Injection Fluorescence Detection

Immunoassay (IA) is a sensitive and selective approach for low level quantitation of drugs. Magnetic separation immunoassays use magnetic beads to facilitate the separation of bound labeled antigens from free antigens in solution. Digoxin was chosen for this study because low level analysis (ng mL−1) in biological samples isrequired, antibodies to digoxin were commercially available and derivatization procedures for fluorescence labeling were well established. A competitive immunoassay format was used in this study. Streptavidin coated magnetic beads were attached to biotinylated anti-digoxin antibodies for the separation. The inhibition curve for off-line magnetic separation immunoassay of digoxin in spiked plasma was characterized and the dynamic range of the curve was 0.25–2.5 ng mL−1. A power fit weighted by the inverse of concentration was found to provide the best fit to the data (r=0.9934). The percent RSDs for the two controls, 0.8 and 2.2 ng mL−1, were 9.95% and 20.62% (n=6) and the percent errors were 11.75% and 22.63% (n=6), respectively. The limit of detection (LOD) in plasma is 0.14 ng mL−1. The dynamic range of the inhibition curve for on-line magnetic separation immunoassay of digoxin was 0.5–15 ng mL−1 of digoxin. A quadratic fit was found to provide the best fit to the data (r=0.9937). The percent RSDs for the two controls, 4.0 and 12 ng mL−1, were 14.1% and 10.7% (n=6) and the percent errors were 5.8% and 3.3% (n=6) from the spiked value, respectively. The LOD was estimated to be 0.44 ng mL−1 (determined as two times the standard deviation of the blank, n=6). The on-line method has the advantages of being relatively easy to automate in the continuous flow mode and is adaptable for use in conjunction with HPLC separations.

Homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin

A novel homogeneous electrogenerated chemiluminescence immunoassay (ECLIA) for the determination of small hapten was developed. As a model system, digoxin was investigated while luminol served as luminescence label and BSA served as carrier protein. Digoxin was indirectly heavily labeled with luminol through BSA to form luminol–BSA–digoxin conjugate. The immunocomplex of luminol–BSA–digoxin conjugate with anti-digoxin antibody underwent less ECL reaction than luminol–BSA–digoxin conjugate after immunoreaction took place. Two immunoassay formats, directly homogeneous immunodetection for anti-digoxin antibody and competitive immunoassay for digoxin, were proposed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration was determined in the range from 1/50,000 to 1/2000 dilution. The ECL intensity versus digoxin concentration was linear in the range from 5.0×10 −10 to 3.0×10 −8 g ml −1. The detection limit was 2.8×10 −10 g ml −1. The relative standard derivation for 1.0×10 −9 g ml −1 was 5.1%. The proposed method has been applied to assay digoxin in human control serum with satisfactory results.