Tetra-primer ARMS-PCR combined with GoldMag lateral flow assay for genotyping: simultaneous visual detection of both alleles.

Rapid and simple detection of single nucleotide polymorphism (SNP) is vital for individualized diagnosis and eventual treatment in the current clinical setting. In this study, we developed a tetra-primer ARMS-PCR combined lateral flow assay (T-ARMS-PCR-LFA) method for simultaneous visual detection of two alleles. By using four primers labeled with digoxin, biotin and Cy5 separately in one PCR reaction, the amplified allele-specific products could be captured by streptavidin and the anti-Cy5 antibody on two separated test lines of a LFA strip, which allows the presentation of both alleles within the single LFA strip. Both DNA and whole blood can be used as templates in this genotyping method in which the whole detection process is completed within 75 minutes. The performance assay of T-ARMS-PCR-LFA demonstrates the accuracy, specificity and sensitivity of this method. One hundred human whole blood samples were used for MTHFR C677T genotyping in T-ARMS-PCR-LFA. The concordance rate of the results detected was up to 100% when compared with that of the sequencing results. Collectively, this newly developed method is highly applicable for SNP screening in clinical practices.

Physiologically based pharmacokinetic modelling of acute digoxin toxicity and the effect of digoxin-specific antibody fragments.

CONTEXT: Recommended doses of digoxin-specific antibody fragments (digoxin-Fab) for treatment of acute digoxin poisoning are pharmacokinetically unsubstantiated and theoretically excessive. Physiologically based pharmacokinetic (PBPK) modelling creates clinical simulations which are closely related to physiological and pharmacokinetic behaviour. This paper details the formulation of a PBPK model of digoxin and explores its use as a simulation tool for acute digoxin toxicity and its management. MATERIALS AND METHODS: A PBPK model of digoxin was constructed and validated for acute digoxin poisoning management by comparing simulations with observed individual acute overdose patients. These simulations were compared with standard two-compartment PK model simulations. RESULTS: PBPK model simulations showed good agreement with post-absorption plasma concentrations of digoxin measured in 6 acute overdose patients. PBPK predictions were accurate to 1.5-fold or less of observed clinical values, proving to be more accurate than two-compartment simulations of the same patients which produced up to a 4.9-fold change. CONCLUSIONS: Compared to conventional two-compartment modelling, PBPK modelling is superior in generating realistic simulations of acute digoxin toxicity and the response to digoxin-Fab. Simulation capacity provides realistic, continuous data which has the potential to substantiate alternative, less expensive, and safer digoxin-Fab dosing strategies for the treatment of acute digoxin toxicity.

Antibody-Bridged Beacon for Homogeneous Detection of Small Molecules.

In conventional competitive immunoassays for small molecules (SM), antibodies are either immobilized to solid phases or labeled with magnetic particles or probes. The former involves laborious blocking and washing steps, whereas the latter requires complicated labeling and purification steps. To circumvent these limitations, we describe here a new type of molecular beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of SM without any immobilization or labeling of the antibody. The AbB is formed by the binding of an antibody to a pair of SM-labeled oligonucleotide probes that each comprise a stem sequence conjugated by either a fluorophore or a quencher. Competitive binding of the SM target to the antibody destructs the stem-loop structure of AbB, restoring the quenched fluorescence. A minimum binding energy of stem sequences is required for efficient formation of the desired stem-loop structure of AbB. A systematic study of the impact of stem sequences on the fluorescence background and quenching efficiency provided useful benchmarks, e.g., binding energy of -11 kcal/mol, for the construction of AbB. The optimized AbB showed fast signal responses, as demonstrated in the analyses of two small molecule targets, biotin and digoxin. Low nanomolar limits of detection were achieved. The novel AbB strategy, along with the guidelines established for the construction and application of AbB, offers a promising approach for homogeneous detection of small molecules, obviating immobilization or labeling of antibodies as required by other competitive immunoassays.

Clinical judgement perplexed by initially undisclosed use of herbal medicine and unexpected cross-reactivity of immunoassay.

We report a case of symptomatic bradycardia caused by consumption of a Chinese herbal medicine which was initially undisclosed to the attending emergency physician. The scientific name of the herb is Panax japonicus. Electrocardiogram revealed sinus bradycardia. Laboratory tests were normal except for the detection of a high serum digoxin level. Further interrogation of the patient eventually disclosed ingestion of the herb which, however, did not contain any digoxin. Other active ingredients in the herb include various types of ginsenoside. These are digoxin-like substances that had caused the observed false-positive detection of digoxin by fluorescence polarization immunoassay due to cross-reactivity. Our case-report provides an important insight about a blind-spot in the field of laboratory medicine (clinical pathology), namely, the false positive detection of digoxin due to crossreactivity in the immunoassay when we come across digoxin-like substances in clinical scenarios, which has barely received attention in the medical literature. It also conveys a clear educational message that with full understanding of the laboratory methodology and its mechanistic rationale there are actually some tricks-of-the-trade that allow us to optimize the specificity of the biochemical tests and the treatment of digoxin-like substances overdose.

Successful use of digoxin-specific immune Fab in the treatment of severe Nerium oleander toxicosis in a dog.

OBJECTIVE: To describe a case in which digoxin-specific immune Fab was used successfully in a dog with severe oleander toxicosis secondary to ingesting plant material. CASE SUMMARY: A 6-year-old intact female Rhodesian Ridgeback mixed breed dog was presented for severe oleander toxicosis and was refractory to all antiarrhythmic therapies and supportive care. Digoxin-specific immune Fab was successful in treating this dog. The dog recovered but suffered ischemic injuries, the long-term effects of which are unknown. NEW OR UNIQUE INFORMATION PROVIDED: This report describes the successful use of digoxin-specific immune Fab in the treatment of oleander toxicosis in a dog, which has not previously been published in veterinary literature. Oleander poisoning can be associated with permanent cardiac arrhythmias due to the ischemic damage.

Comfrey herbal remedy causing second-degree heart block: do not be outfoxed by digitalis.

A previously well woman aged 63 years presents to the emergency department with vomiting, palpitations and 3 presyncopal episodes. She had no previous medical or cardiac history, with the patient stating that she tried a herbal remedy of boiled comfrey leaves for insomnia 18 hours before arrival to the department. Her ECG showed multiple abnormalities, including bradycardia, second-degree atrioventricular node block, Mobitz Type 2, a shortened QT interval, downsloping ST depression and presence of U waves. After viewing the images of comfrey and foxglove, it highlighted the possibility of mistaken ingestion of Digitalis, containing the organic forms of cardiac glycosides, such as digoxin and digitoxin. Raised serum digoxin levels confirmed this. The patient was haemodynamically stable, and given digoxin-binding antibodies. After 5 days of cardiac monitoring, her ECG returned to normal rhythm, and she was discharged home.

Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-ß). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-ß antibody to the target protein. Polyclonal anti-NGF-ß antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-ß, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-ß. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Quantitative evaluation of biological reaction kinetics in confined nanospaces.

Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis.

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.

Recurrence of digoxin toxicity following treatment with digoxin immune fab in a patient with renal impairment.

In patients with chronic digoxin toxicity, especially in the presence of renal impairment, a prolonged duration of continuous monitoring is required with consideration given to further doses of immune fab if necessary for re-emergence of toxicity.

Production and characterization of recombinant scFv against digoxin by phage display technology.

The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of treated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).

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.