Anti-digoxin Antibody Research Articles

Efficacy and effectiveness of anti-digoxin antibodies in chronic digoxin poisonings from the DORA study (ATOM-1)

abstractContext: We hypothesized that in chronic digoxin toxicity, anti-digoxin antibodies (Fab) would be efficacious in binding digoxin, but this may not translate into improved clinical outcomes. Objective: This study aims to investigate changes in free digoxin concentrations and clinical effects on heart rate and potassium concentrations in chronic digoxin poisoning when anti-digoxin Fab are given. Materials and methods: This is a prospective observational study. Patients were recruited if they have been treated with anti-digoxin Fab for chronic digoxin poisoning. Data was entered into a standardised prospective form, supplemented with medical records. Their serum or plasma was collected, analysed for free and bound digoxin and free anti-digoxin Fab concentrations. Results: From September 2013 to February 2015, 36 patients (median age, 78 years; 22 females) were recruited from 18 hospitals. Median heart rate (HR) was 49 beats/min. Initial median digoxin and potassium concentrations were 4.7 nmol/L (3.6 μg/L) (range: 2.3–11.2 nmol/L) and 5.3 mmol/L (range: 2.9–9.2 mmol/L) respectively. Beta-blockers (n = 18), calcium antagonists (n = 6), spironolactone and/or angiotensin blocking agents (n = 24) were also used concomitantly. Renal impairment and gastrointestinal symptoms were present in 31 (86%) and 22 (63%) patients respectively. Five patients died from conditions unrelated to digoxin toxicity. Median change in HR was 8 beats/min post-Fab with no effect on blood pressure; they were 4, 10 and 17 beats/min for the 1, 2 and ≥3 vials of anti-digoxin Fab groups respectively. Concomitant treatments with potassium lowering agents (12/36) and inotropic drugs (7/36) were used. Gastrointestinal effects resolved in all 22 patients. The median decrease for potassium was 0.3 mmol/L. Digoxin concentration reduced from 3.8 to 0 nmol/L post-Fab. There was a rebound observed in the free digoxin concentration in 25 patients but none had associated clinical deterioration. Conclusions: One to two vials of anti-digoxin Fab initially bound all free digoxin confirming Fab efficacy. However, this was associated with only a moderate improvement in HR and potassium, suggesting bradyarrhythmia and hyperkalaemia may be from other co-morbidities.

Direct detection of microRNA-126 at a femtomolar level using a glassy carbon electrode modified with chitosan, graphene sheets, and a poly(amidoamine) dendrimer composite with gold and silver nanoclusters

We describe a strategy for the determination of microRNA-126 (miRNA-126) that is based on the use of a glassy carbon electrode modified with (a) carboxy-terminated generation 3.5 poly(amidoamine) (PAMAM) dendrimer; (b) gold and silver nanoclusters, and (c) a chitosan-graphen composite. A peptide nucleic acid (PNA) was immobilized on the surface and serves as a receptor to hybridize miRNA-126. Subsequently, a digoxin labeled signal DNA is specifically recognized by the PNA. The analytical signal was finally generated by adding anti-digoxin antibody labeled with horse radish peroxidase. The biosensor was characterized by differential pulse voltammetry. A linear current-concentration relationship is found for miRNA-126 at a working voltage of 208 mV in the 1.0 fM to 10 nM concentration range. The limit of detection is 0.79 fM (at an S/N of 3). This biosensor displays good reproducibility, stability and selectivity.

Synthesis and Characterization of Core-shell Au Fe Oxide Nanocomposites and Their Application for Detecting Immunological Interaction

Gold (Au) Fe oxide core-shell nanostructure is a major class of nanoscale materials that has attracted considerable attention due to its biotechnological and biomedical applications and properties. Here we synthesized magnetite nanoparticles (MNPs), an average size of 10-12 nm in diameter, using the chemical co-precipitation of Fe2(+) and Fe3(+) in strong alkaline material. Then, an Au-coated Fe oxide core-shell nanocomposite with an average diameter of 18-20 nm was produced in the presence of sodium citrate as the reducing agent. The core-shell structure and the presence of the Fe and Au phases have been confirmed by transmission electron microscopy (TEM), x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The Au-coated Fe oxide NPs can be used as a carrier in digoxin immunoassays. The concentration of anti-digoxin antibody (Ab) immobilized on the NPs was 30 μg/mg. In order to validate the quality of the Au-coated Fe oxide NPs as immunoassay carriers, an immunoassay system was developed. The relative amount of anti-digoxin Ab immobilized on the NPs was determined by enzyme linked immunosorbent assay (ELISA). Immobilization studies with Ab demonstrated that the Au Fe oxide core-shell nanostructure can be used to immobilize Ab, making them valuable for biomedical and biological applications.

PKP-007 Analysis of patients with digoxin intoxication admitted to hospital as emergencies

BackgroundDigoxin, the oldest cardiovascular drug, has been used for the treatment of heart failure and in frequency control strategies in atrial fibrillation (AF) patients. American College of Cardiology/American Heart Association...

Pathophysiologically-Relevant Levels of Endogenous Cardiotonic Steroids Inhibit the Cardiac Na/K ATPase and Activate ERK1/2 Hypertrophic Signaling In Vivo and In Vitro

Type-4 cardiorenal syndrome patients develop heart failure as result of kidney failure. While the mechanism of cardiac dysfunction is unknown, elevated levels of circulating endogenous Na/K ATPase inhibitors (NKAi) are detected in patients and animals with kidney failure. We investigated the pathophysiological consequences of NKAi elevation in rat myocytes and hearts of animals with chronic kidney disease (CKD).Methods: Intracellular Na concentration ([Nai]) was measured using fluorescent ratio-imaging microscopy and ERK1/2 phosphorylation by western blotting, in adult rat ventricular myocytes. Cardiac Na/K ATPase (NKA) activity was measured using biochemical NKA assays. CKD was induced in rats via 5/6 partial-nephrectomy (PN) or in mice via folic-acid administration (FA). Creatinine serum levels were increased in both models, with kidney dysfunction (and cardiac hypertrophy) being more severe in PN model.Results: Treatment of field-stimulated rat cardiac myocytes with bufalin (10nM) or ouabain (100nM) significantly increased [Nai] by 1.2±0.3 and 1.9±0.2mM, respectively, whereas, concentrations of marinobuganenin below 1μM did not. Furthermore, NKAi activated hypertrophic signaling cascades (assessed by ERK1/2 phosphorylation) with a potency order of bufalin (EC50=70nM)>ouabain (EC50=93nM)> marinobuganenin (EC50=6.4μM). Despite small but significant increases in NKA alpha-1/2 expression, NKA activity in hearts of animals with kidney dysfunction was reduced by 45±19% in PN (6 weeks post-PN) and 34±9% in FA animals (12 weeks post FA-injection). NKA inhibition was reversed by incubation with a polyclonal anti-Digoxin antibody (binds most structurally-related NKAi), confirming involvement of NKAi.Conclusions: Pathophysiologically-relevant concentrations of bufalin and ouabain, but not marinobufagenin, raise [Nai] and activate hypertrophic signaling pathways in the heart, thus contributing to Na and diastolic Ca overload and hypertrophy, during CKD.

Passive Immunization Against Marinobufagenin Attenuates Renal Fibrosis and Improves Renal Function in Experimental Renal Disease

We have shown that the cardiotonic steroid marinobufagenin (MBG) is elevated in clinical and experimental renal disease, and significantly contributes to the development of experimental uremic cardiomyopathy induced by removal of five-sixths of the kidney (5/6 nephrectomy; PNx) in the rat. We have demonstrated that both active and passive immunization against MBG with an anti-MBG monoclonal antibody (mAb 3E9) significantly attenuated cardiac fibrosis following PNx. In the present study we sought to determine whether the use of mAb 3E9 could improve renal function following PNx. Sprague-Dawley rats were treated with either mAb 3E9 or with DigiFab (an affinity-purified anti-digoxin antibody formerly named Digibind) during the fourth week after PNx. Sham-operated animals and PNx animals treated with an IgG antibody served as controls. Plasma, urine, and renal tissue were collected at the completion of the study to determine the effects of antibody treatment on renal function. In PNx rats, treatments with mAb 3E9 and DigiFab, respectively, significantly reduced plasma creatinine, improved creatinine clearance, and reduced proteinuria below the values of these three measures in IgG-treated PNx controls. Additionally, treatment with mAb 3E9 and DigiFab significantly reduced renal fibrosis as measured with Western blotting and Sirius red/Fast green staining. Passive immunization against MBG significantly improved renal function and markedly reduced renal fibrosis following the experimental induction of renal disease. The work in the study reported here adds to a growing body of knowledge implicating MBG in the development of chronic renal disease. Passive immunization against cardiotonic steroids may serve as a promising treatment for chronic renal disease.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.

Digoxin antibody fragment, antigen binding (Fab), treatment of preeclampsia in women with endogenous digitalis-like factor: a secondary analysis of the DEEP Trial

Endogenous digitalis-like factors (EDLFs) are elevated in women with preeclampsia, and the use of an anti-digoxin antibody Fab (DIF) in women with preeclampsia who were remote from term reduced maternal blood pressure and preserved renal function. The objective was to determine whether DIF treatment in women with severe preeclampsia in association with positive EDLFs in maternal serum improves maternal-perinatal outcomes. This was a planned secondary analysis from a randomized, placebo-controlled, double-blind study of DIF in women with severe preeclampsia with positive EDLF status that was managed expectantly between 23 weeks 5 days and 34 weeks' gestation (19 women received placebo, and 17 women received DIF). Primary outcome variables were a change in creatinine clearance and the use of antihypertensives. Secondary outcomes were maternal and perinatal complications. Women with positive EDLFs who received DIF had an attenuated decline in creatinine clearance from baseline compared with placebo (-4.5 ± 12.9 vs -53.2 ± 12.6 mL/min; P = .005). In this same group, the use of antihypertensives (the other primary outcome) was lower but not significantly so (41% vs 63%; P = .12). However, women who were treated with DIF had a lower rate of pulmonary edema (1/17 vs 6/19 women; P = .035) and lower rates of neonatal intraventricular hemorrhage (DIF: 0/17 women vs placebo: 5/19 women; P = .015). In women with severe preeclampsia who were remote from term who were EDLF positive, the use of DIF was associated with improved maternal and neonatal outcome. These findings suggest the need for a large multicenter trial that would evaluate the benefits of DIF in the treatment of women with severe preeclampsia who are remote from term and with positive EDLF status.

Anti-Digoxin Fab Variants Generated by Phage Display

Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is narrow, with effect dosage very close to the toxic dosage. To counteract the toxic effect, polyclonal Fab fragments are commercially available. Our study is based on a monoclonal anti-digoxin antibody, which would provide a product with a specific potency and more precise dosage for the detoxification of patients under digoxin treatment. Phage display technology was used to select variants with high affinity. From an anti-digoxin hybridoma, RNA was extracted for subsequent cDNA synthesis. Specific primers were used for the LC and Fd amplifications, then cloned sequentially in a phagemid vector (pComb3X) for the combinatorial Fab library construction. Clones were selected for their ability to bind to digoxin-BSA. The presence of light and heavy chains was checked, randomly selected clones then sequenced and induced to produce soluble Fabs, and subsequently analyzed for anti-digoxin expression. Out of ten clones randomly chosen, six resulted positive expression of the product. The sequencing of these revealed two identical clones and one presenting a pseudogene in the LC. Four clones presenting variations in the framework1 showed binding to digoxin-BSA by ELISA and western blotting. The specific binding was further confirmed by Biacore(®), which allowed ranking of the clones. The development of these clones allowed the selection of variants with higher affinity than the original version.

Increasing Evidence for and Regulation of a Human Placental Endogenous Digitalis-Like Factor

Endogenous digitalis-like factors (EDLFs) appear to be hypertensiogenic and increased in the serum and placenta of women with preeclampsia (PE), a complication of pregnancy. Digibind, an anti-digoxin antibody Fab fragment, reverses in vitro effects of EDLF and in vivo features of PE. We used Digibind in a radioimmunoassay to measure EDLF and compared this to a bio-functional assay of EDLF with good agreement. These methods confirmed that human placenta was a source of EDLF, synthesizing and releasing EDLF into the media of cultured human placental tissue. Ketoconazole, a steroid synthesis inhibitor, and 17-OH progesterone, a possible substrate of steroid synthesis, were shown to inhibit or increase EDLF release respectively, suggesting overlap of synthetic pathways. Abnormalities of PE such as placental hypoxia, increased reactive oxygen species and increased pro-inflammatory cytokines were demonstrated to increase placental EDLF release. These findings strongly support placental production of EDLF with increased release due to features of PE.

Visual detection of gene mutations based on isothermal strand-displacement polymerase reaction and lateral flow strip

Here, we describe a simple and sensitive approach for visual detection of gene mutations based on isothermal strand-displacement polymerase reactions (ISDPR) and lateral flow strip (LFS). The concept was first demonstrated by detecting the R156H-mutant gene of keratin 10 in Epidermolytic hyperkeratosis (EHK). In the presence of biotin-modified hairpin DNA and digoxin-modified primer, the R156H-mutant DNA triggered the ISDPR to produce numerous digoxin- and biotin-attached duplex DNA products. The product was detected on the LFS through dual immunoreactions (anti-digoxin antibody on the gold nanoparticle (Au-NP) and digoxin on the duplex, anti-biotin antibody on the LFS test zone and biotin on the duplex). The accumulation of Au-NPs produced the characteristic red band, enabling visual detection of the mutant gene without instrumentation. After systematic optimization of the ISDPR experimental conditions and the parameters of the assay, the current approach was capable of detecting as low as 1-fM R156H-mutant DNA within 75min without instrumentation. Differentiation of R156H- and R156C-mutant DNA on the R156 mutation site was realized by using fluorescein- and biotin-modified hairpin probes in the ISDPR process. The approach thus provides a simple, sensitive, and low-cost tool for the detection of gene mutations.

Visual detection of Hg 2+ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes

We report a sensitive method for visual detection of mercury ions (II) (Hg 2+) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine–Hg 2+–thymine (T–Hg 2+–T) coordination in the presence of Hg 2+. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg 2+ without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg 2+ and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.

Instantaneous derivatization technology for the simultaneous and homogeneous detection of multiple double-stranded PCR amplicons

Herein we report on the development of instantaneous derivatization technology for the homogeneous and simultaneous detection of multiple PCR amplicons specific to the Hepatitis B Virus (HBV) by using three carriers: magnetic beads, polystyrene beads, and thermo-sensitive poly-N-isopropylacrylamide (PNIP). Briefly, PCR amplicons are labeled with digoxin, biotin or FITC via the modified up-stream primers respectively. After PCR amplification, the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with anti-digoxin antibody, streptavidin or anti-FITC antibody in a single vessel, and then each carrier is separated from the others under different conditions based on their physio-chemical attributes. And then direct CL detection proceeds via the instantaneous derivatization reaction between intrinsic guanine nucleobases and 3,4,5-trimethoxylphenylglyoxal (TMPG). This new protocol directly measures the double-stranded DNA and therefore does not require a denaturing step, thus offering an enhanced sensitivity due to the absence of competitive hybridization, i.e., the detection limit had a 20-fold improvement on the conventional PCR measurement. Additionally, by comparison of previous guanine based detection formats, this protocol is easy to be used for the detection of any guanine containing targets without the use of guanine-free or inosine-substituted capture probes. Overall, the proposed technique takes the advantages of sensitivity, high-speed and cost-effectivity, which provides a promising alternative for the analysis of multiple PCR targets in a variety of clinical, environmental, and biodefense fields.

Interference Between Eplerenone and Digoxin in Fluorescence Polarization Immunoassay, Microparticle Enzyme Immunoassay, and Affinity Column-Mediated Immunoassay

Digitalis-like immunoreactive substances have crossreactivity with antidigoxin antibodies and the interference between digoxin and spironolactone/canrenone has been reported. The structure of eplerenone is similar to that of spironolactone/canrenone. Therefore, we hypothesized that eplerenone might also interfere with the measurement of digoxin by immunoassay. We performed three types of assays (fluorescence polarization immunoassay [FPIA], microparticle enzyme immunoassay [MEIA], and affinity column-mediated immunoassay [ACMIA]) to determine crossreactions between eplerenone and antidigoxin antibodies. Furthermore, we used FPIA, MEIA, and ACMIA to measure the apparent digoxin concentration in mixed solutions of eplerenone (1-100 μg/mL) and digoxin (1-3 ng/mL). In the crossreaction tests, eplerenone was detected as digoxin by FPIA and ACMIA. By FPIA, a known concentration of 1 μg/mL of eplerenone was measured as 0.33 ± 0.11 ng/mL of digoxin (crossreaction rate, 0.03%). By ACMIA, a known concentration of 10 μg/mL of eplerenone was measured as 0.13 ± 0.05 ng/mL of digoxin (crossreaction rate, 0.001%). No crossreaction between eplerenone and digoxin was determined by MEIA. In the interference of eplerenone coadministered with digoxin, the apparent concentration of digoxin was increased in FPIA, but decreased in MEIA and ACMIA. The results suggest that eplerenone crossreacts with antidigoxin antibodies in FPIA, MEIA, and ACMIA, but that the interference of eplerenone might be smaller than that of spironolactone/canrenone.

Ultrasensitive electrochemical detection of nucleic acid based on the isothermal strand-displacement polymerase reaction and enzyme dual amplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPR to produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collection via an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody on the magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidin interaction. The quantification of DNA was realized by square wave voltammetric detection of the enzymatic products with a screen-printed gold electrode. The voltammetric response was proportional to the concentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM. The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.

Availability of antidotes for the treatment of acute poisoning in Queensland public hospitals

To determine the sufficiency of stock levels of 13 antidotes in Queensland hospitals. A self-report survey was sent to 128 Queensland hospitals with acute care facilities. The stock level of the following antidotes was determined: acetylcysteine, anti-digoxin Fab antibodies (digibind), atropine, calcium gluconate, cyanokit, desferrioxamine, flumazenil, glucagon, intravenous ethanol, methylene blue, naloxone, pralidoxime and pyridoxine. Other factors sampled were bed capacity, rural, remote and metropolitan areas classification, use of formal stock reviews by pharmacists or nurses, existence of formal borrowing agreements with other facilities for non-stocked antidotes, distance to the nearest referral hospital and time taken to transfer antidotes from another hospital. Pharmacists or nurses responsible for maintaining antidote stocks in Queensland hospitals. Proportions of hospitals with sufficient antidote stock to treat a 70-kg adult for four or more hours using previously published guidelines. Survey response rate was 73.4%. No hospital had sufficient stock of all 13 antidotes. The proportion of hospitals with sufficient stocks varied from 0% (pyridoxine) to 68.1% (acetylcysteine). Larger hospitals had a higher frequency of sufficient antidote stocks. Only 16% of hospitals claimed to be able to acquire an antidote from another facility within 30 min. Most Queensland hospitals stocked some important antidotes, but few had sufficient stock to treat a 70-kg patient or acquire an antidote within the recommended time frame of 30 min. Specific antidote stocking guidelines might be required for Queensland hospitals. A formalised program for stock rotation with rural facilities should be explored.

Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

In this study, we used a modified enzyme-linked immunosorbent assay (ELISA)-elution technique as a screening tool to select specific elution conditions. We examined 12 different elution conditions for the removal of antibodies from a complex on an ELISA plate; 0.2 mol/l glycine-HCl (pH 2.5), 1.0 mol/l acetic acid (pH 2.5), 25% methanol (pH 2.5) and 3 mol/l NaSCN showed a higher elution efficiency. We conducted affinity chromatography with these four conditions for the purification of anti-digoxin antibodies from hyperimmune sera with a digoxin-specific column using omega-aminoalkyl derivatives of Sepharose 4B, whose elution efficiency was similar to that of ELISA. We also monitored the relative specific activities during elution from the digoxin-specific column. The optimum, general-purpose dissociation reagent for this immunoaffinity system was identified as 25% methanol (pH 2.5) with an elution efficiency and relative specific activity of 88.40% and 62.25%, respectively. The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Emergency step-by-step specific immunotherapy in severe digoxin poisoning: an observational cohort study

To evaluate the efficacy and safety of a step-by-step fixed dose of specific immunotherapy protocol in case of severe digoxin poisoning in an open uncontrolled prospective study. Twenty consecutive patients were admitted because of severe digoxin poisoning. The inclusion criteria were: digoxin overdose and either life-threatening arrhythmia; high-degree atrioventricular block, ventricular arrhythmia, or bradycardia less than 50 bpm and hyperkalaemia (>5.5 mmol/l). A two-step protocol of antidigoxin antibodies treatment was carried out. At admission, every patient received two vials of specific Fab-fragments. If after 1 h following infusion ECG signs regressed, no more treatment was given. If ECG signs did not regress, patients were given two more vials. At inclusion and 6 h after immunotherapy, clinical (cardiac rhythm, ECG records) and biological (serum digoxin concentration, potassium) findings were recorded. Patients had a median (interquartile range) age of 83 (75-90) years. Four patients had acute poisoning and 16 chronic overdoses. Eleven patients showed ventricular arrhythmia, and five had high-degree atrioventricular block. Seventy percent of the patients needed only the first step. Significant decreases were observed in the number of cardiac dysrhythmia (16 vs. three patients), in the median (interquartile range) of serum digoxin concentration [5 microg/l (3.8-6.2) vs. 0.4 microg/l (0.3-2.2)] and in serum potassium [4.6 mmol/l (4.1-5.5) vs. 3.85 mmol/l (3.7-4.55)] before and after immunotherapy. The digoxin-related mortality was 5%. This protocol of step-by-step digoxin-specific immunotherapy seems to be as effective as the equimolar treatment, and there was significant cost reduction in case of acute poisoning.

A MIP-based flow-through fluoroimmunosensor as an alternative to immunosensors for the determination of digoxin in serum samples

This work reports a comparative study of two automated flow-through fluorosensors for the determination of digoxin in serum samples: an immunosensor with an anti-digoxin polyclonal antibody as the reactive phase permanently immobilised on controlled-pore glass and a sensor with a selective reaction system based on a methacrylic molecularly imprinted polymer (MIP) synthesised by bulk polymerisation. The variables affecting the sensitivity and dynamic range of the sensors (e.g. the carrier and elution solutions, flow rates, pH and reagent concentrations) were optimized, and the binding characteristics of their reactive phases were compared in a competitive fluorescent assay. Digoxin was reproducibly determined by both sensors at the milligram per litre level (detection limit = 1.20 x 10(-3) mg L(-1) and RSD = 4-7% for the immunosensor; detection limit = 1.7 x 10(-5) mg L(-1) and RSD = 1-2% for the MIP sensor). No cross-reactivity with digoxin-related compounds was seen for either sensor at a digoxin/interferent ratio of 1:100. The lifetime of the immunosensor was about 50 immunoassays; its shelf life, when unused, is about 3 months. The lifetime of the MIP sensor was over 18 months. Both sensors were used to determine the digoxin concentration of human serum samples with satisfactory results.

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension

Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.