Richard O’Kennedy

Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities

The current outbreak of Zika virus (ZIKV) infection has been associated with an apparent increased risk of congenital microcephaly. We describe a case of a pregnant woman and her fetus infected with ZIKV during the 11th gestational week. The fetal head circumference decreased from the 47th percentile to the 24th percentile between 16 and 20 weeks of gestation. ZIKV RNA was identified in maternal serum at 16 and 21 weeks of gestation. At 19 and 20 weeks of gestation, substantial brain abnormalities were detected on ultrasonography and magnetic resonance imaging (MRI) without the presence of microcephaly or intracranial calcifications. On postmortem analysis of the fetal brain, diffuse cerebral cortical thinning, high ZIKV RNA loads, and viral particles were detected, and ZIKV was subsequently isolated.

Advances in biosensors for detection of pathogens in food and water

Abstract While most microbes play an important role in nature, certain potentially harmful microbes can contaminate food and water, and cause a plethora of infectious diseases in both animals and humans. Conventional methods for detecting microbial contamination have primarily relied on time-consuming enrichment steps, followed by biochemical identification, having a total assay time of up to 1 week in certain cases. Over the last decade, a great deal of research has focused on the development of biological sensors for the detection of micro-organisms, allowing rapid and “real-time” identification. This paper reviews some of the most commonly used biosensor systems based on their transducer properties, which include surface plasmon resonance (SPR), amperometric, potentiometric, and acoustic wave sensors and their applications for the detection of pathogens in food and water. It also highlights some of the limitations of applying biosensors for the detection of pathogens, such as sensitivity, cost and the need for sample pre-treatment.

Mastitis detection: current trends and future perspectives

Bovine mastitis, the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. Traditionally, methods of detection have included estimation of somatic cell counts, an indication of inflammation, measurement of biomarkers associated with the onset of the disease (e.g. the enzymes N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase) and identification of the causative microorganisms, which often involves culturing methods. These methods have their limitations and there is a need for new rapid, sensitive and reliable assays. Recently, significant advances in the identification of nucleic acid markers and other novel biomarkers and the development of sensor-based platforms have taken place. These novel strategies have shown promise, and their advantages over the conventional tests are discussed.

Antibody production, design and use for biosensor-based applications

Currently, the reliable detection and quantification of a multitude of different analytes is crucial in many applications and settings. Biosensors have revolutionised diagnostics for use in point-of-care testing (POC), the detection of food and environmental contaminants, biological warfare agents, illicit drugs and human/animal disease markers. Antibodies continue to play a pivotal role in many sensor devices due to their exquisite specificity for their cognate antigens. In this review current biosensor platforms employing antibodies for molecular recognition are briefly described. The use of molecular biological techniques for the generation and improvement of antibodies is critically examined. Such recombinant antibodies possess improved attributes for use in biosensor development in terms of design, stability, affinity and specificity.

A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications

Artificial manipulation of antibody genes has facilitated the production of several unique recombinant antibody formats, which have highly important therapeutic and biotechnological applications. Although bispecific antibodies (bsAbs) are not new, they are coming to the forefront as our knowledge of the potential efficacy of antibody-based therapeutics expands. The next generation of bsAbs is developing due to significant improvements in recombinant antibody technologies. This review focuses on recent advances with a particular focus on improvements in format and design that are contributing to the resurgence of bsAbs, and in particular, on innovative structures applicable to next generation point-of-care (POC) devices with applicability to low resource environments.

Development of a High Sensitivity Rapid Sandwich ELISA Procedure and Its Comparison with the Conventional Approach

A highly sensitive and rapid sandwich enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of human fetuin A/AHSG (alpha2-HS-glycoprotein), a specific biomarker for hepatocellular carcinoma and atherosclerosis. Anti-human fetuin A antibody was immobilized on aminopropyltriethoxysilane-mediated amine-functionalized microtiter plates using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide-based heterobifunctional cross-linking. The analytical sensitivity of the developed assay was 39 pg/mL, compared to 625 pg/mL for the conventional assay. The generic nature of the developed procedure was demonstrated by performing human fetuin A assays on different polymeric matrixes, i.e., polystyrene, poly(methyl methacrylate), and polycyclo-olefin (Zeonex), in a modified microtiter plate format. Thus, the newly developed procedure has considerable advantages over the existing method.

Affinity chromatography as a tool for antibody purification

The global antibody market has grown exponentially due to increasing applications in research, diagnostics and therapy. Antibodies are present in complex matrices (e.g. serum, milk, egg yolk, fermentation broth or plant-derived extracts). This has led to the need for development of novel platforms for purification of large quantities of antibody with defined clinical and performance requirements. However, the choice of method is strictly limited by the manufacturing cost and the quality of the end product required. Affinity chromatography is one of the most extensively used methods for antibody purification, due to its high selectivity and rapidity. Its effectiveness is largely based on the binding characteristics of the required antibody and the ligand used for antibody capture. The approaches used for antibody purification are critically examined with the aim of providing the reader with the principles and practical insights required to understand the intricacies of the procedures. Affinity support matrices and ligands for affinity chromatography are discussed, including their relevant underlying principles of use, their potential value and their performance in purifying different types of antibodies, along with a list of commercially available alternatives. Furthermore, the principal factors influencing purification procedures at various stages are highlighted. Practical considerations for development and/or optimizations of efficient antibody-purification protocols are suggested.

Polyreactivity as an acquired artefact, rather than a physiologic property, of antibodies: evidence that monoreactive antibodies may gain the ability to bind to multiple antigens after exposure to low pH

Evidence is presented that monoreactive antibodies exposed to low pH may acquire the ability to bind to multiple antigens. M11, a murine, monoclonal, IgM(K) anti-goat IgG (GIgG) was purified from a hybridoma supernatant by elution at low pH from an anti-mu-Sepharose 4B affinity column. By measuring the specific antiGIgG activities and the affinity constants for the interactions of M11, pre- and post-affinity-purification, with GIgG, M11 was shown to be monoreactive before purification. Quite unexpectedly, however, the affinity-purified M11 reacted extensively with size-fractionated liver proteins when tested in an immunoblot, clearly indicating that it was polyreactive. It was concluded that the exposure to low pH had altered the M11 binding-site so as to allow it to bind to many different proteins. This phenomena provides an alternative basis for interpreting the polyreactivity observed following affinity-purification.

Nanobiotechnologies for the detection and reduction of pathogens.

Advances in the manipulation of nanomaterials has permitted the development of nanobiotechnology with enhanced sensitivities and improved response times. Low levels of infection of the major pathogens require the need for sensitive detection platforms and the properties of nanomaterials make them suitable for the development of assays with enhanced sensitivity, improved response time and increased portability. Nanobiotechnologies focusing on the key requirements of signal amplification and pre-concentration for the development of sensitive assays for food-borne pathogen detection in food matrices will be described and evaluated. The potential that exists for the use of nanomaterials as antimicrobial agents will also be examined.

Determination of 20 coccidiostats in egg and avian muscle tissue using ultra high performance liquid chromatography-tandem mass spectrometry.

A quantitative, comprehensive multiresidue method which includes 20 coccidiostat residues has been developed. The method described uses a simple one-step liquid extraction with acetonitrile to isolate analytes from both the polyether ionophore and chemical classes of coccidiostats. Subsequent to a further concentration step, samples were analysed via UHPLC-MS/MS. The method was validated according to the Commission Decision 2002/657/EEC in egg and avian muscle. The method permitted quantitative confirmation for 13 compounds below target concentrations, and screening for a further 7 compounds. Within-laboratory repeatability gave accuracy values in the range of 68-129%, while reproducibility ranged between 75 and 123%. Calibration ranges were typically 1-50 μg kg⁻¹, although higher ranges were used for dinitrocarbanilide, imidocarb and toltrazuril residues. A regression coefficient (R²) value of greater than 0.98 was obtained for all analytes. Precision results ranged from 2.3 to 19.7% CV for egg and from 2.6 to 23.6% CV in muscle. CCα was in the range from 1.13 μg kg⁻¹ (clopidol) to 179 μg kg⁻¹ (lasalocid) in egg. In muscle, CCα ranged from 2.25 μg kg⁻¹ (aprinocid) to 4579 μg kg⁻¹ (dinitrocarbanilide). CCβ was from 1.29 μg kg⁻¹ (clopidol) to 209 μg kg⁻¹ (lasalocid) in egg, and 2.58 μg kg⁻¹ (arprinocid) to 6060 μg kg⁻¹ (dinitrocarbanilide) in muscle. Limits of quantification were 1 μg kg⁻¹ for all compounds, except imidocarb and dinitrocarbanilide (10 μg kg⁻¹), and toltrazuril and metabolites (50 μg kg⁻¹).